Soluble urokinase plasminogen activator receptor in patients with abdominal aortic aneurysm

Introduction

In the present study we investigated the impact of soluble urokinase plasminogen activator receptor (suPAR) as a biomarker in patients with abdominal aortic aneurysm (AAA) in relation to conventional inflammatory markers, aneurysm size, and rupture.

Methods

suPAR and conventional inflammatory markers were measured in 119 patients with AAA and 36 controls without aneurysm matched by age, gender and smoking habit.

Results

The results support earlier studies suggesting a state of activated inflammatory response in patients with nonruptured AAA as expressed by elevated CRP and IL-6 compared with the controls. In contrast, suPAR showed similar levels in patients with nonruptured AAA compared with the controls. Unexpectedly, all follow-up patients (n = 16) have significant (p < 0.001) elevated suPAR levels three years postoperatively compared preoperatively.

Conclusions

suPAR does not seem to be a useful biomarker in the AAA disease. The role of the postoperative elevation of suPAR needs to be further elucidated.     

Neuroprotection by urokinase plasminogen activator in the hippocampus

Tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which are both used for thrombolytic treatment of acute ischemic stroke, are serine proteases that convert plasminogen to active plasmin. Although recent experimental evidences have raised controversy about the neurotoxic versus neuroprotective roles of tPA in acute brain injury, uPA remains unexplored in this context. In this study, we evaluated the effect of uPA on neuronal death in the hippocampus of mice after kainate-induced seizures. In the normal brain, uPA was localized to both nuclei and cytosol of neurons. Following severe kainate-induced seizures, uPA completely disappeared in degenerating neurons, whereas uPA-expressing astrocytes substantially increased, suggesting reactive astrogliosis. uPA-knockout mice were more vulnerable to kainate-induced neuronal death than wild-type mice. Consistent with this, inhibition of uPA by intracerebral injection of the uPA inhibitor UK122 increased the level of neuronal death. In contrast, prior administration of recombinant uPA significantly attenuated neuronal death. Collectively, these results indicate that uPA renders neurons resistant to kainate-induced excitotoxicity. Moreover, recombinant uPA suppressed cell death in primary cultures of hippocampal neurons exposed to H2O2, zinc, or various excitotoxins, suggesting that uPA protects against neuronal injuries mediated by the glutamate receptor, or by oxidation- or zinc-induced death signaling pathways. Considering that tPA may facilitate neurodegeneration in acute brain injury, we suggest that uPA, as a neuroprotectant, might be beneficial for the treatment of acute brain injuries such as ischemic stroke.     

Differences in the activation rates of plasminogen by tissue plasminogen activator and urokinase

The activation of a native form of plasminogen (Glu-plg) by tissue plasminogen activator(t-PA) was enhanced when the plasma was clotted by the addition of thrombin or thrombin plus Ca++. Cross-linking of fibrin in the clotted plasma did not inhibit the fibrin-associated enhancement of the activation of plasminogen by t-PA. When fibrinolysis induced by t-PA in the clotted plasma was measured using enzyme immunoassay, lysis of non cross-linked fibrin in the clotted plasma was faster than lysis of cross-linked fibrin, however such decrease in the extent of fibrinolysis was observed in cross-linked fibrin even in the absence of alpha 2antiplasmin (alpha 2AP) in a purified system. When Glu- or Lys-plg (modified plg) was activated by t-PA, the presence of fibrin enhanced significantly the extent of activation of both Glu- and Lys-plg, but the activation of Glu-plg by urokinase (UK) was enhanced in the presence of fibrin. The activation of Lys-plg by UK was rather inhibited in the presence of fibrin.     

Aprotinin inhibits urokinase but not tissue-type plasminogen activator

The protease inhibitor, aprotinin, has been examined for its ability to inhibit urokinase and tissue-type plasminogen activators at pH 7.4 in assays utilizing pyroGlu-Gly-Arg-p-nitroanilide and H-D-Ile-Pro-Arg-p-nitroanilide as substrates, respectively. Aprotinin inhibited both two-chain low molecular weight urokinase and the high molecular weight form of the enzyme in a competitive manner with a similar Ki (27 μM). There was no observable inhibition of tissue-type plasminogen activators at aprotinin concentrations up to 500 uM. These findings suggest that sensitivity to inhibition by aprotinin could be used to distinguish tissue-type and urokinase-type plasminogen activators.     

Regulation of the urokinase receptor by its plasminogen activator.

There is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to assess the role of UK as a modulator of its receptor. GEO colonic cells, which secrete relatively low levels of UK (congruent to 0.1 nM/72 h per 10(6) cells) and display approximately 10(4) receptors per cell, 10% of which are "tagged" with the endogenous plasminogen activator (PA), was selected for the study. A 90% reduction in the specific binding of radioactive DFP-UK was observed for cells cultivated in the presence of two-chain (TC) UK (Mr 55,000). This only partly reflected occupation of the receptors with UK supplied in the culture medium, since the specific binding of the radioligand was still reduced by 60% after an acid pretreatment, which dissociates receptor-bound UK. The reduction in radioactive DFP-UK binding to cells treated with high molecular weight UK, either in the single or two-chain form, was both concentration and time dependent. Maximum reductions (70%) were achieved by treatment of the cells for 24 h with 1 nM of the plasminogen activator. In contrast, low molecular weight UK, which lacks part of the UK A chain, had no effect on ligand binding. Attenuation of radioactive DFP-UK binding to UK treated GEO cells was a consequence of a 60% reduction in the number of binding sites. Treatment of GEO cells with an antibody, which blocks the binding of endogenous UK to its receptor, augmented radioactive DFP-UK binding by two-fold. These data indicate that for one colonic cell line, at least, UK down-regulates its own binding site subsequent to it being bound to the receptor.     

Immobilization of plasminogen activator, urokinase, on nylon

Urokinase (UK), a fibrinolytic enzyme activator purified from human material was immobilized on nylon using different procedures. One was a modified method of immobilization of antigen or antibody initially carried out by Edelman and others in 1971 (Procedure I). The other was our newly devised method (Procedure II) (Sugitachi et al. 1976). Major specificities of the immobilized UK are as follows: 1. The UK revealed properties of a plasminogen activator and the optimum pH of the immobilized UK was between 7.2 and 7.4, these values being in good parallel with that of soluble UK. The immobilized UK maintained a stable fibrinolytic activity after long-term preservation and heat-treatment. 2. As the fibrinolytic activity of immobilized UK was found to be inhibited by the antiplasmin in human plasma, an antiplasmin inhibitor was immobilized on the nylon together with the UK. The antiplasmin activity was to some extent prevented using this procedure. 3. Nylon tubes immobilized with UK and antiplasmin inhibitor were used for thrombotic coagulation studies carried out according to the method of Chandler. Thrombus formation time (TFT) of UK-immobilized tubes was 30 min, while that of the non-treated tubes was no longer than 10 min.     

The urokinase type of plasminogen activator in cancer of digestive tracts

The amounts of urokinase (UK) antigen and tissue plasminogen activator (t-PA) antigen were determined in plasma, urine and tissues of patients with cancer of digestive tracts. Urinary levels of UK, but not those of t-PA increased in patients with cancer, and generally decreased after the removal of cancer by operation. Urinary UK levels kept increasing in patients with recurrence of cancer or with metastasis into liver or peritoneum. Plasma levels of t-PA, but not those of UK decreased in patients with cancer. When the amounts of UK were compared in cancer tissues and their adjacent normal mucosal layer, cancer tissues always had higher levels of UK, but t-PA levels were same between tumor tissues and normal mucosa. The results suggest that the type of plasminogen activator was UK-type in cancer of digestive tracts.     

Roles of urokinase type plasminogen activator in a brain stab wound

Urokinase type plasminogen activator (uPA) may influence brain pathophysiology after injury. We studied disruption of the blood-brain barrier (BBB) and changes in the vasculature after a brain stab wound in uPA-deficient, uPA receptor-deficient, and PA inhibitor-1 (PAI-1) deficient mice. The extravasation of immunoglobulin was greater in PAI-1 deficient mice; less pronounced in uPA-deficient mice; similar to controls in uPA receptor-deficient mice. Vasculatures in the wound proliferated in PAI-1 deficient mice. Our study shows that uPA affects BBB disruption. PA enhances angiogenesis after brain injury.     

Absence of synergism between tissue-type plasminogen activator and urokinase on plasminogen activation rate in plasma

Effects of tissue-type plasminogen activator (t-PA), urokinase(u-PA) and their combinations on plasminogen activation rate (PAR) in plasma, were investigated. T-PA and u-PA over concentrations range of 10 U/ml to 50 U/ml induced a linear, concentration dependent increase in PAR. Combinations of t-PA and u-PA in ratios of 3/1,1/1 and 1/3 induced additive but not synergistic effect in the activation of plasminogen. We conclude, therefore, that t-PA and u-PA do not act synergistically in the activation of plasminogen in plasma .     

Dissolution of thrombi by tissue plasminogen activator, urokinase and streptokinase in an artificial circulating system

The ability of porcine tissue plasminogen activator, urokinase and streptokinase to dissolve whole blood clots has been compared in an artificial circulating system, the Chandler loop. On a molar basis the tissue activator was more effective in lysing both partially and totally crosslinked thrombi than urokinase or streptokinase. The latter drugs required 30–50 times higher concentrations to achieve a fibrinolytic response, similar to that of tissue activator. As demonstrated by analysis of fibrinogen, ₂-antiplasmin and plasmin-₂-antiplasmin complex there was no activation of plasminogen in plasma by the tissue activator in the absence of fibrin. Both urokinase and streptokinase gave under the same conditions rise to a significant dose dependent fibrinogenolysis. In the presence of fibrin, tissue activator induced a weak fibrinogenolysis (about 20%) in plasma, whereas both urokinase and streptokinase caused a much higher degree of fibrinogenolysis (50 and 60 % respectively).     

The plasminogen activator and esterase activities of the two forms of urokinase

High and low mol. wt. urokinase (UK) purified to homogeneity by affinity chromatography, and partially purified commercial preparations with varying ratios of high to low mol. wt. forms were assayed for plasminogen activator activity by: physiologic clot lysis time using varying amounts of native and degraded plasminogen; plasminogen activator activity by protamine assay; and esterase activity on the synthetic peptide ester N-Ac-Gly-Lys-O-Me (AGLMe); as well as by active-site titration using p-nitrophenyl-p′-guanidino-benzoate (NPGB). The molar activity of both high and low mol. wt. UK by the three rate assays was about 1 × 10¹³ IU/mole active site and was not affected by molecular size or low mol. wt. impurities. Depending on the particular UK preparation and the type of plasminogen, one or more impurities may affect the fibrinolytic assay at limiting substrate concentrations. Gel filtration or affinity chromatography abolishes the effect. NPGB titration provides another rapid and simple means of estimating UK activity and relating all three rate assays to molar activities. Correlation of the results of protamine and AGLMe assays with those of the fibrinolytic assays was generally good, except for the tissue culture UK which showed at least 25% higher values by both the protamine and AGLMe assays. The protamine assay proved a useful alternative to fibrinolytic and synthetic substrate.     

Importance of plasminogen activator inhibitor type 1 (PAI-1) for preventing single chain urokinase plasminogen activator (scu-PA) conversion into two chain urokinase plasminogen activator (tcu-PA) in plasma in vitro.

We have studied the effects of PAI-1 on the conversion of scu-PA into tcu-PA in vitro in plasma containing or not a 125I-fibrin clot by determining tcu-PA activity on S2444. Two preparations of PAI-1 have been used, a fraction of medium conditioned with the monkey Vero cells (Vero-Prep), the antiurokinase activity of which is inhibited at 83% by anti PAI-1 IgG, or purified human PAI-1 from HT 1080 fibrosarcoma cells. Scu-PA purified from human kidney cells has been treated with diisopropylfluorophosphate before use. In plasma, conversion of scu-PA into the tc form is accelerated by addition of anti PAI-1 IgG. In plasma containing a clot, generation of tcu-PA, is considerably delayed after addition of the Vero-Prep or human PAI-1. Clot lysis is also decreased but to a lesser extent than it would be expected from the level of tcu-PA activity. Addition of anti PAI-1 antibodies shortens the lag phase before tcu-PA appears and moderatly accelerates clot lysis. These results demonstrate the importance of PAI-1 for the stability of scu-PA in plasma in vitro by delaying its conversion into tcu-PA.     

Expression of plasminogen activator inhibitors type 1 and type 3 and urokinase plasminogen activator protein and mRNA in breast cancer

BACKGROUND: The urokinase plasminogen activator (uPA) system has been involved in cancer cell invasion and in metastasis. uPA activity is controlled by its principal inhibitor, the PA inhibitor type-1 (PAI-1), but it can also be inhibited by PAI-3. Increased levels of uPA and PAI-1 are known to be associated with a poor prognosis in breast cancer. To our knowledge this is the first study of the expression and role of PAI-3 in human breast cancer tissue. MATERIALS AND METHODS: Protein and mRNA levels were evaluated for uPA, PAI-1 and PAI-3 in breast cancer tissues from 70 different patients. The localization of antigen and mRNA of these proteins was studied by immunohistochemistry and in situ hybridization, respectively. RESULTS: No significant differences were observed for PAI-3 mRNA or protein levels between the nodal status groups or the different post-surgical tumor-node-metastasis (pTNM) stages. However, uPA and PAI-1 mRNA and antigen levels significantly increased at the pTNM stage and in node-positive patients. PAI-3 antigen levels were significantly higher in early relapse-free patients, whereas PAI-1 antigen levels were significantly higher in patients who suffered a relapse. PAI-3 protein and mRNA were localized in stromal cells. PAI-1 and uPA protein were detected in cancer, endothelial and stromal cells and their mRNA mainly in stromal cells. CONCLUSIONS: Our results indicate that PAI-3 is expressed in human breast cancer tissues, and that elevated levels of PAI-3 could be a positive prognostic factor in this disease. A potential mechanism for the contribution of PAI-3 to a positive long-term outcome may involve suppression of tumor invasion through protease inhibition in stroma.     

Evaluation of fibrinolytic capacity in plasma during thrombolytic therapy with single (scu-PA) or two-chain urokinase type plasminogen activator (tcu-PA) by a combined assay system for urokinase type plasminogen activator antigen and function

A combined assay for urokinase type plasminogen activator (u-PA) activity and antigen determination in plasma samples is described. This assay is based on binding of u-PA to an antibody immobilized on a microtiter plate followed by determination of the enzymatic activity of the bound u-PA. Thereafter bound u-PA antigen can be quantified by means of a specific peroxidase labelled monoclonal antibody against u-PA. By use of this assay system u-PA activity and antigen can be determined with lower detection limits of 0.08 IU/ml and 1.0 ng/ml, respectively, and intraassay as well as interassay coefficients of variation of 10% and 12% for activity and 5% and 7% for antigen determinations, respectively. Normal plasma levels of u-PA antigen could be determined to be 1.88 ng/ml +/- 0.61. Furthermore, this assay system allows specific quantification of u-PA antigen and activity during thrombolytic therapy.     

The concentration of tissue plasminogen activator and urokinase in plasma and tissues of patients with ovarian and uterine tumors

The concentrations of tissue plasminogen activator (t-PA) and urokinase (UK) were measured in the plasma of patients with benign or malignant ovarian and uterine tumors. Plasma levels of t-PA were higher in patients with malignant ovarian and uterine tumors than in patients with benign tumors. Plasma UK levels were, however, not different between patients with ovarian and uterine tumors (benign or malignant) and normal persons. The concentrations of t-PA and UK of tissues of uterine myoma were lower than those in normal uterine muscular layer. UK levels of tissues of endometrial and cervical cancers were significantly higher than those in normal endometrium and uterine cervix, whereas t-PA levels were not different between these tumors compared with those in normal uterine tissues.