Induction of tissue plasminogen activator in differentiated NG108-15 cells

Plasminogen activators may facilitate neurite outgrowth and neuronal migration in the developing nervous system. The expression of tissue plasminogen activator by NG108-15 neuroblastoma grown under a variety of conditions has been explored. High tissue plasminogen mRNA expression correlates with growth conditions which induce morphological differentiation and neurite outgrowth; however, NG108-15 cells grown in suspension with dibutyryl-cAMP also show a high level of tissue plasminogen activator expression.     

Importance of the interaction between plasminogen and fibrin for plasminogen activation by tissue-type plasminogen activator

The potentiating effect of fibrin monomer on plasminogen activation by tissue-type plasminogen activator is much more important with lys-plasminogen than with mini-plasminogen (which lacks the high affinity lysine-binding site important for binding to fibrin). Furthermore, this potentiating effect is totally abolished when lys-plasminogen is eluted from fibrin by the addition of 1 mM epsilon-amino caproic acid. Binding does however not seem to be the only condition required since it was found that fragment D is a much stronger potentiator of the activation of plasminogen by tissue-type plasminogen activator than fragment E although plasminogen binds to both fragment D and fragment E. Furthermore, fragment E has the same effect on the activation of lys-and mini-plasminogen by tissue-type plasminogen activator. Therefore, it is suggested that binding of plasminogen to fibrin involves a conformational change in the plasminogen molecule, facilitating its activation by tissue-type plasminogen activator.     

水蛭提取液对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物和纤溶酶原激活剂抑制物1的影响

目的探讨水蛭提取液(HEL)对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物(tPA)、纤溶酶原激活剂抑制物1(PAI-1)的影响。方法建立大鼠大脑皮质微血管内皮细胞培养实验模型。MTT法筛选HEL的有效浓度。检测培养上清液的tPA、PAI-1含量与活性变化,RT-PCR检测经HEL治疗组与生理盐水对照组处理后的微血管内皮细胞tPA与PAI-1的表达,免疫组化检测两组微血管内皮细胞tPA的表达。结果 HEL在一定浓度范围内(0.25～1mg/μl)可促进微血管内皮细胞的生长,有剂量依赖关系(P<0.05)。HEL治疗组较生理盐水对照组能促进培养的大鼠脑皮质微血管内皮细胞分泌tPA,同时提高其活性,促进tPA mRNA的表达及tPA免疫活性表达,且呈剂量依赖性表达增强(P<0.01)。结论 HEL在体外能激活内源性纤溶系统。     

Poly-lysines as modifiers of one- and two-chain tissue-type plasminogen activator activity

Poly-L-lysine and certain mixed polymers of L-lysine and other amino acids modify the activity of one- and two-chain tissue-type plasminogen activator (t-PA) towards its substrates. In particular the rate of plasminogen activation in the presence of optimal poly-L-lysine concentrations, is increased by approximately 100-fold. In contrast, activity towards a small synthetic substrate is inhibited by 85%. These effects are observed with both the one- and two-chain forms of t-PA. The use of poly-L-lysines in a coupled assay system optimised for t-PA and plasmin activities allfys the reproducible assay of t-PA at the 10^-12 to 10^-13 molar level.     

Quantitation of tissue-type plasminogen activator in human endothelial cell cultures by use of an enzyme immunoassay

Endothelial cells from human umbilical cord were cultured to study plasminogen activator synthesis and secretion. Since simultaneous production of plasminogen activator inhibitor(s) prevented detection of plasminogen activators by use of fibrinolytic assays, an enzyme immunoassay for tissue-type plasminogen activator (t-PA) was developed. In this assay, t-PA in test samples was adsorbed onto microtiter plates coated with rabbit antibody against t-PA and then quantitated by successive incubation with goat antibody against t-PA and enzyme labeled rabbit antibody against goat IgG. The sensitivity of the assay was found to be 1 ng t-PA/ml. In the absence of serum, arterial and venous endothelial cells continuously produce t-PA antigen during a 24 h period, reaching a level of 5.1 +/- 2.5 ng t-PA/ml (n = 8). In serum containing (20%) medium, 9.3 +/- 6.0 ng t-PA/ml (n = 17) was produced during this period (0.1 - 0.2 ml medium per cm2 confluent cells). It is concluded that the enzyme immunoassay is a useful method for quantitating t-PA secretion by endothelial cells in the presence of proteinase inhibitors.     

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.     

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 .     

Urokinase‐type plasminogen activator modulates mammalian circadian clock phase regulation in tissue‐type plasminogen activator knockout mice

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue‐type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA^?/?; B6.129S2‐Plat^tm1Mlg/J) mice, and identify urokinase‐type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA^?/? mice entrain to a light‐dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA^?/? mice take significantly longer to entrain than C57BL/6J wild‐type (WT) mice. SCN brain slices from tPA^?/? mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose‐dependency. Pre‐treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor‐1 (PAI‐1), inhibits glutamate‐induced phase delays in tPA^?/? slices. Selective inhibition of uPA with UK122 prevents glutamate‐induced phase resetting in tPA^?/? but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA^?/? slices. Casein‐plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA^?/? SCN. Finally, tPA^?/? SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.     

Influence of exogenous and endogenous tissue-type plasminogen activator on the lysability of clots in a plasma milieu in vitro

The fibrinolytic potential of tissue-type plasminogen activator (t-PA) either incorporated in a clot (endogenous) or added to the surrounding plasma (exogenous), was studied in an in vitro system consisting of 125I-labeled human plasma clots (200 microliters) immersed in human plasma (2 ml). Clot lysis was measured as a function of endogenous t-PA concentration (in the absence of added exogenous t-PA), as a function of exogenous t-PA concentration (without added endogenous t-PA) and as a function of the same concentration of both endogenous and exogenous t-PA. Equivalent clot lysis was obtained with a 2 to 4 times lower concentration of endogenous t-PA as compared to exogenous t-PA, corresponding to a 20 to 40 times smaller total amount of endogenous versus exogenous t-PA. Fifty percent lysis in 5 hrs was obtained with about 5 IU/ml of endogenous t-PA or with 10 IU/ml of exogenous t-PA. The presence of both exogenous (10 IU/ml) and endogenous (5 IU/ml) t-PA resulted in 50 percent lysis in 1.5 hrs, indicating that t-PA incorporated in a thrombus contributes significantly to its lysis by exogenous t-PA. Similar results were obtained with plasma obtained after 10 min of venous occlusion in seven healthy subjects. Spontaneous clot lysis within 5 hrs was only observed with post-occlusion clots in pre- or post- occlusion plasma in two subjects in whom the t-PA level rose to 10-15 IU t-PA/ml. In the five other subjects with post-occlusion t-PA levels below 2 IU/ml, no clot lysis was observed within 24 hrs. The influence of the fast-acting inhibitor of t-PA on clot lysability by endogenous or exogenous t-PA was investigated by immersing clots prepared from normal or inhibitor-rich plasma (endogenous inhibitor) in normal or inhibitor-rich plasma (exogenous inhibitor). Exogenous t-PA inhibitor efficiently neutralizes clot lysis by both exogenous and endogenous t-PA. Endogenous t-PA inhibitor, however, efficiently neutralizes endogenous t-PA but has little influence on clot lysis by exogenous t-PA. These findings indicate that t-PA inhibitor is not concentrated into a clot and that t-PA inhibitor in plasma efficiently neutralizes t-PA incorporated in a clot. alpha 2-Antiplasmin depleted plasma clots were more susceptible to lysis by both endogenous and exogenous t-PA than normal clots.(ABSTRACT TRUNCATED AT 400 WORDS)     

尿激酶型纤维蛋白酶原激活物受体的反义基因片断抑制胶质瘤u25i细胞的体外侵袭

目的：研究尿激酶型纤维蛋白酶原激活物受体（uPAR）的反义基因片断对胶质瘤细胞株u251体外侵袭能力的影响。方法：脂质体介导寡核苷酸转染培养的u251胶质瘤细胞，用RT—PCR、原位杂交和免疫组化方法分别检测相关基因和蛋白的表达，Boyden小室模型研究对肿瘤细胞侵袭能力的影响。结果：uPAR反义寡核苷酸作用u251胶质瘤细胞后相关基因的mRNA及蛋白的表达明显减弱，细胞的体外侵袭能力被明显抑制。结论：uPAR的反义寡核苷酸片断能有效地抑制胶质瘤细胞u251相关基因的表达，并能抑制胶质瘤细胞的体外侵袭能力。     

尿激酶型纤维蛋白酶原激活物受体的反义基因片断抑制胶质瘤u251细胞的体外侵袭

目的:研究尿激酶型纤维蛋白酶原激活物受体(uPAR)的反义基因片断对胶质瘤细胞株u251体外侵袭能力的影响。方法:脂质体介导寡核苷酸转染培养的u251胶质瘤细胞,用RT-PCR、原位杂交和免疫组化方法分别检测相关基因和蛋白的表达,Boyden小室模型研究对肿瘤细胞侵袭能力的影响。结果:uPAR反义寡核苷酸作用u251胶质瘤细胞后相关基因的mRNA及蛋白的表达明显减弱,细胞的体外侵袭能力被明显抑制。结论:uPAR的反义寡核苷酸片断能有效地抑制胶质瘤细胞u251相关基因的表达,并能抑制胶质瘤细胞的体外侵袭能力。     

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.     

Tissue plasminogen activator in human plasma measured by radioimmunoassay

A radioimmunoassay (RIA) has been set up using purified human melanoma tissue plasminogen activator and a specific antiserum raised against it. Concentration of antigen in citrated plasma of resting human subjects was found to be 7.1 ± 1.6 ng/ml (mean ± S.D.) while the lower limit of sensitivity of the RIA was 2 ng/ml. After venous occlusion or DDAVP infusion the levels of antigen were invariably elevated and a significant increase in the proportion of antigen having fibrin binding properties was observed. Decay studies $\text{in vitro}$ and gel filtration of post-stimulus plasma indicated that the RIA detects active and inactive forms of antigen and the possible nature of the inactive antigen is discussed.     

Induction of plasminogen activator in a human diploid fibroblast cell line (MRC-5) by conditions which cause induction of interferon: the role of calcium ions

The human diploid fibroblast cell line, MRC-5, derived from embryo lung tissue produced only small quantities of plasminogen activator (PA) when harvested using a standard nutrient medium (Eagle's Minimal Essential Medium, MEM). Use of a schedule designed to induce high concentrations of fibroblast interferon in these cells also resulted in production of considerably enhanced levels of PA. The kinetics of PA production differed from those of interferon production; specifically, PA was produced for at least 6 days following induction despite the toxicity of the inducers whereas interferon synthesis continued for only 1 day. Further investigation of the induction conditions for PA revealed that double-stranded RNA which was absolutely required for interferon production was not required for induction of PA. Indeed, the stimulus for enhancement of PA production appeared to be solely an elevated concentration of calcium ions in the extracellular medium. The possible physiological relevance of this induction of PA by elevated concentrations of calcium ions is discussed.     

重组组织型纤溶酶原激活剂静脉溶栓治疗后循环脑梗死的疗效及安全性评价

目的探讨rt-PA(重组组织型纤溶酶原激活剂)静脉溶栓治疗后循环脑梗死的疗效及安全性。方法选取我院2013-09—2015-09收治的62例后循环脑梗死患者,依据治疗方法不同分为常规组与研究组各31例。常规组予以常规治疗,研究组予以rt-PA溶栓治疗。比较2组治疗前后NIHSS(神经功能缺失评分量表)评分,观察2组临床效果、继发性脑出血率及病死率、血管再闭塞发生情况。结果与常规组比较,研究组治疗后NIHSS评分较低,差异有统计学意义(P0.05);研究组总有效率(93.5%)高于常规组(64.5%),差异有统计学意义(P0.05);2组继发性脑出血、血管再闭塞发生率及病死率比较,差异均无统计学意义(P0.05)。结论 rt-PA溶栓治疗后循环脑梗死,可显著改善患者神经功能,临床效果较为显著,且安全性较高,在临床治疗中具有重要意义。     

Induction of synthesis of plasminogen activator inhibitor type-1 by tissue-type plasminogen activator in human hepatic and endothelial cells

Plasminogen activator inhibitor type-1 (PAI-1) can modify fibrinolytic activity in vitro and in vivo. The present study was performed to determine whether pharmacologic concentrations of tissue-type plasminogen activator (t-PA) can initiate negative feedback by stimulating PAI-1 synthesis. In both human hepatoma cells (Hep G2) and human umbilical vein endothelial cells (HUVEC), t-PA increased the total concentrations and appearance of newly synthesized protein in conditioned media of free PAI-1 and PAI-1 complexed with t-PA in a dose and time dependent fashion judging from results after immunoprecipitation of metabolically labeled PAI-1. The t-PA effect was not attributable simply to release of stored or matrix-bound PAI-1. In HUVEC, Northern blot analyses indicated that t-PA increased steady-state levels of PAI-1 mRNA two-fold. In contrast PAI-1 mRNA expression was not increased in Hep G2 cells. Thus, mechanisms of stimulation appeared to differ in the two cell lines. The results obtained are consistent with the hypothesis that increased PAI-1 synthesis and secretion in response to t-PA may limit or attenuate fibrinolysis locally or systemically in vivo.     

uPA和KDR在髓母细胞瘤中的表达及意义

目的 研究尿激酶型纤溶酶原激活剂（urokinase-type plasminogen activator,uPA)和KDR（vascular endothelia growth factor receptor-2)在髓母细胞瘤中的表达及临床意义。方法 应用免疫组化LSAB法检测50例髓母细胞瘤中uPA和KDR的表达，结合临床随访，使用Cox回归统计分析和Spearman相关分析。结果 uPA和KDR的染色定位于肿瘤细胞和血管内皮细胞。Cox归分析显示uPA和KDR是影响生存时间的独立的预后因子，它与预后存在高度负相关关系。Spearman相关分析显示uPA和KDR存在正相关关系。结论 uPA和KDR可能是预测髓母细胞瘤预后的独立的预后因子。uPA和KDR两者正相关关系。     

Monoclonal antibody to human tissue plasminogen activator

Hybridoma producing a monoclonal antibody IgG₁ to one-chain tissue plasminogen activator (t-PA) derived from human melanoma cells was obtained by fusion of mouse myeloma cells (SP-1) and spleen cells of mice previously immunized with purified t-PA. The monoclonal antibody reacted only with t-PA derived from the human melanoma cells (Bowes), and not with plasminogen activator purified from porcine heart or from human urine. The monoclonal antibody obtained from mouse ascites demonstrated 50 times stronger antibody activity than that of polyclonal antibody obtained from mouse serum. The monoclonal antibody bound t-PA firmly, inhibited the fibrinolytic activity of t-PA, but did not inhibit the amidolytic activity of t-PA completely. The fib-χin-binding ability of t-PA was not inhibited. The binding of monoclonal antibody to the non-reduced form of t-PA did not differ from that to the reduced form of t-PA.