Levels of tissue-type plasminogen activator and plasminogen activator inhibitor 1 in disseminated intravascular coagulation syndrome

Since disseminated intravascular coagulation (DIC) may directly reflect the abnormal regulation of the fibrinolytic system by endothelial cells, we have measured the levels of tissue-type plasminogen activator (t-PA), type 1 PA inhibitor (PAI-1) and t-PA . PAI-1 complex which is formed as a result of interaction on the two factors, in the plasma of patients with DIC (n = 51) and healthy controls (n = 42). Antigens of t-PA, PAI-1 and t-PA . PAI-1 complex were significantly increased in the DIC plasma (36.4 +/- 25.1, 106.8 +/- 54.7 and 46.6 +/- 34.5 ng/ml, respectively) compared with those in normal plasma (8.5 +/- 4.3, 54.4 +/- 21.2 and 8.6 +/- 3.5 ng/ml, respectively). The molar ratio of t-PA to PAI-1 was much higher in the DIC plasma (1:3) than in normal plasma (1:6), which caused enhancement of the whole fibrinolytic activity in the DIC plasma. These changes resulted in significant consumption of plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI) and a significant increase of plasmin . alpha 2-PI complex (PPI) and D-dimer. These results suggest that t-PA and its specific inhibitor PAI-1 both of which are secreted from endothelial cells into blood, play an important role on the progress of DIC.     

组织型纤溶酶原激活物基因的DNA改组

目的：探索利用DNA改组（DNAshuffling)技术，获得更高活性tPA的可能性。方法：以人，恒河猴及大白鼠tPAcDNA为一组基因，进行tPA的DNA改组（DNAfamily shuffling)。以改组后构建的tPA多样性文库转染CHO细胞并进行克隆和筛选。结果：得到了两株有意义的克隆；t9和t17，其中t9克隆表达的tPA活性略高于人tPA，初步的比活性测定结果表明，活性约提高4倍。t17克隆表达的tPA虽然有88个氨基酸的缺失，但仍表现出与人tPA相同的活性，两株克隆经测序证明，为改组后的基因，其序列以人和恒河猴的tPAcDNA序列为主，少数序列来源于大白鼠tPAcDNA。结论：这一探索性结果将为后续几轮的tPADNA改组探明道路，为最终从改组后tPA多样性基因库中筛选到比较理想的重组体打下基础。     

Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured monkey Sertoli cells

Sertoli cells play a central role in the control and maintenanceof spermatogenesis. Isolated Sertoli cells of mouse and rattestes have been shown to secrete plasminogen activator (PA)and a plasminogen activator inhibitor type-1 (PAI-1) in culture.In this study, we have investigated the hormonal regulationof PA and PAI-1 activities in cultured monkey Sertoli cells.Sertoli cells (5x10⁵ cells/well) isolated from infant rhesusmonkey testes were preincubated at 35°C for 16 h in 24-wellplates precoated with poly(D-lysine) (5 µg/cm²) in 0.5ml McCoy's 5a medium containing 5% of fetal calf serum and furtherincubated for 48 h in 0.5 ml serum-free medium with or withoutvarious hormones or other compounds. PA as well as PAI-1 activitiesin the conditioned media were assayed by fibrin overlay andreverse fibrin autography techniques respectively. The Sertolicells in vitro secreted only tissue-type PA (tPA), no detectableamount of urokinase-type PA (uPA) could be observed. MonkeySertoli cells were also capable of secreting PAI-1. Immunocytochemicalstudies indicated that both tPA and PAI-1 positive staininglocalized in the Sertoli cells, spermatids and residual bodiesof the seminiferous epithelium; Northern blot analysis furtherconfirmed the presence of both tPA and PAI-1 mRNA in monkeySertoli cells. Addition of follicle-stimulating hormone (FSH)or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generatingagents and gonadotrophin-releasing hormone (GnRH) agonist orphorbol ester (PMA) to the cell culture significantly increasedtPA activity. PAI-1 activity in the culture was also enhancedby these reagents except 8-bromo-dibutyryl-cAMP, forskolin and3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPAactivity, whereas decreased PAI-1 activity, implying that neutralizationof PAI-1 activity by the high level of tPA in the conditionedmedia may occur. These data suggest that increased intracellularsignals which activate protein kinase A (PKA), or protein kinaseC (PKC) can modulate Sertoli cell tPA and PAI-1 activities.The concomitant induction of PA and PAI-1 by the same reagentsin the Sertoli cells may reflect a finely tuned regulatory mechanismin which PAI-1 could limit the excession of the proteolysis. plasminogen activator inhibitor type-1/Rhesus monkey/Sertoli cells/tissue-type plasminogen activator     

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).     

Amyloid-beta-stimulated plasminogen activation by tissue-type plasminogen activator results in processing of neuroendocrine factors

Alzheimer's disease brain is characterized by the abundant presence of amyloid deposits. Accumulation of the major constituent of these deposits, amyloid-beta (Abeta), has been associated with decreased neurotransmission, increased neuronal cell death, and with cognitive decline. The mechanisms underlying these phenomena have not yet been fully elucidated. We have previously shown that amyloid peptides like Abeta bind tissue-type plasminogen activator (tPA) and cause enhanced plasmin production. Here we describe the identification of five major neuronal cell-produced Abeta-associated proteins and how Abeta-stimulated plasmin formation affects their processing. These five proteins are all neuroendocrine factors (NEFs): chromogranins A, B and C; truncated chromogranin B; and VGF. Plasminogen caused processing of Abeta-bound (but not soluble) tPA, chromogranin B and VGF and the degradation products were released from Abeta. Processing of the neuroendocrine factors was dependent on tPA as it was largely abrogated in tPA-/- cells or in the presence of a specific tPA-inhibitor. If plasmin indeed produces NEF-derived peptides in vivo, some of these peptides may have biological activity, for instance in regulating neurotransmitter release that may affect the pathology of Alzheimer's disease.     

Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by Rickettsia conorii- and Rickettsia rickettsii-infected cultured endothelial cells.

Hemostasis abnormalities have been described in patients with Mediterranean spotted fever and Rocky Mountain spotted fever. Evidence of the activation of the fibrinolytic system has been obtained in both diseases. After experimental Rocky Mountain spotted fever, an elevated level of fibrinogen was found in parallel with the activation of the fibrinolytic system and transient elevation of the tissue-type plasminogen activator. Later protein is mainly synthesized by endothelial cells. The ability to culture human endothelial cells in vitro provides a unique system to study the secretion of tissue-type plasminogen activator and of plasminogen activator inhibitor after rickettsial infection. Human vascular endothelial cells derived from the umbilical vein, when infected with Rickettsia conorii or Rickettsia rickettsii, secreted as much tissue-type plasminogen activator as control cells. The activity of plasminogen activator inhibitor however, was higher in the supernatants of infected cells than in those of control cells. This rickettsia-induced imbalance of the tissue-type plasminogen activator-inhibitor pair was a very early event after in vitro infection. The involvement of this system during Mediterranean spotted fever and Rocky Mountain spotted fever remains to be demonstrated.     

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.     

The possible role of tissue-type plasminogen activator and the plasminogen system in the pathogenesis of major depression

Major depressive disorder (MDD) is one of the most common psychiatric illnesses with an unknown etiology. Evidence from animal and human studies has suggested that brain-derived neurotrophic factor (BDNF) function may be implicated in the pathogenesis of MDD. Tissue-type plasminogen activator (tPA) is a highly specific serine proteinase that catalyses the generation of zymogen plasminogen from the proteinase plasmin. Recent studies have found that the proteolytic cleavage of proBDNF, a BDNF precursor, to BDNF by the plasmin represents a mechanism by which the direction of BDNF action is controlled. Furthermore, studies using mice deficient in tPA has demonstrated that tPA is important for the stress reaction, a common precipitating factor for MDD. A study of the serum levels of the plasminogen activator inhibitor-1 (PAI-1), the major inhibitor of tPA, found that women with MDD had a higher PAI-1 concentration than normal controls. From these findings, it is proposed that the tPA/plasminogen system may play a role in the pathogenesis of MDD. Attempts to confirm the tPA/plasminogen hypothesis may lead to new directions in the study of the pathogenesis of MDD and the development of a novel intervention of this disorder.     

Differing roles for urokinase and tissue-type plasminogen activator in collagen-induced arthritis

The plasminogen activators, urokinase PA (u-PA) and tissue-type PA (t-PA), are believed to play important roles in inflammatory cell infiltration, fibrin deposition, and joint destruction associated with rheumatoid arthritis; however, their precise roles in such processes, particularly u-PA, have yet to be defined. Using gene-deficient mice we examined the relative contribution of the PAs to the chronic systemic collagen-induced arthritis model. Based on clinical and histological assessments, u-PA-/- mice developed significantly milder disease and t-PA-/- mice more severe disease compared with the relevant wild-type mice. Fibrin deposition within joints paralleled disease severity and was particularly pronounced in t-PA-/- mice. Likewise, cytokine levels in the synovium reflected the severity of disease, with interleukin-1beta levels in particular being lower in u-PA-/- mice and increased in t-PA-/- mice. The antibody response to type II collagen was normal in both knockouts; however, T cells from u-PA-/- mice had a reduced proliferative response and produced less interferon-gamma on antigen stimulation in vitro. These results indicate that the major effect of u-PA in the collagen-induced arthritis model is deleterious, whereas that of t-PA is protective. Our data highlight the complexities of PA function, and suggest that approaches either to target u-PA or to enhance local t-PA activity in joints may be of therapeutic benefit in rheumatoid arthritis.     

组织型纤溶酶原激活物单克隆抗体研制及其特性鉴定

目的:应用t-PA单克隆抗体建立ELISA法检测t-PA含量以及研究t-PA功能和结构的关系.方法:运用杂交瘤技术研制5株t-PA单克隆抗体,进行较系统的免疫特性的鉴定.结果:5株单抗特异性高,与u-PA、PLG、Fg、Fb、BSA均无交叉反应;亲和力强,1H4＞3C10＞5H10＞4E6＞4C6;腹水效价5×10-6～1×10-7;免疫球蛋白亚类为IgG1和IgG2a;5株抗体中,3C10和1H4可明显抑制t-PA活性,而5H10、4E6、4C6则对t-PA活性无明显影响.结论:为进一步应用这些单抗作为研究手段提供了基础.     

组织型纤溶酶原激活剂在移植血管桥再狭窄动物血管的差异表达

目的研究组织型纤溶酶原激活剂（t—PA／PLAT）在移植血管桥再狭窄动物血管的差异表达。方法通过兔双侧颈动脉进行动脉桥和静脉桥的移植,形成双侧移植血管桥再狭窄动物模型。应用免疫组化检测t-PA在动物模型动脉桥、静脉桥的表达并进行比较。结果血管桥移植前,t-PA在实验动物颈动脉和颈静脉的表达差异无统计学意义（P〉0．05）;血管桥移植后,t-PA在动脉桥的表达明显高于静脉桥（P〈0．05）,于16周时达到高峰[（32．34±4．74）％比（16．74±3．14）％],以后随时间延长而出现表达减少（P〈0．05）。结论t-PA在术后早期对血管桥具有保护作用,其表达的高低与术后血管桥再狭窄关系密切。     

Effect of recombinant tissue-type plasminogen activator on ten platelet membrane glycoproteins

Recombinant tissue-type plasminogen activator (rt-PA) did not modify densities of nine platelet membrane glycoproteins: collagen receptor subunit GP Ia; fibrinogen receptor GP IIb IIIa; thrombospondin receptor GP IV; von Willebrand factor receptor GP Ib and associated GP IX; thrombospondin; activation glycoprotein GMP 140; vitronectin receptor subunit VNR α and GP Ha. rt-PA induced slight decrease of GP 24 (CD9) density on platelet membrane. Antigen densities were determined after incubation of platelet-rich plasma with therapeutic doses of rt-PA (from 0.5-4 μg/ml) by a quantitative cytofluorometric method. Addition of fibrin did not modify the effect of rt-PA. These results suggest that incubation of platelet-rich plasma with therapeutic doses of rt-PA neither modify glycoproteins involved in platelet adhesion and aggregation nor markedly activate platelets.     

Endocrinology and Paracrinology: Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured monkey Sertoli cells

Sertoli cells play a central role in the control and maintenanceof spermatogenesis. Isolated Sertoli cells of mouse and rattestes have been shown to secrete plasminogen activator (PA)and a plasminogen activator inhibitor type-1 (PAI-1) in culture.In this study, we have investigated the hormonal regulationof PA and PAI-1 activities in cultured monkey Sertoli cells.Sertoli cells (5x105 cells/well) isolated from infant rhesusmonkey testes were preincubated at 35°C for 16 h in 24-wellplates precoated with poly(D-lysine) (5 µg/cm²) in 0.5ml McCoy's 5a medium containing 5% of fetal calf serum and furtherincubated for 48 h in 0.5 ml serum-free medium with or withoutvarious hormones or other compounds. PA as well as PAI-1 activitiesin the conditioned media were assayed by fibrin overlay andreverse fibrin autography techniques respectively. The Sertolicells in vitro secreted only tissue-type PA (tPA), no detectableamount of urokinase-type PA (uPA) could be observed. MonkeySertoli cells were also capable of secreting PAI-1. Immunocytochemicalstudies indicated that both tPA and PAI-1 positive staininglocalized in the Sertoli cells, spermatids and residual bodiesof the seminiferous epithelium; Northern blot analysis furtherconfirmed the presence of both tPA and PAI-1 mRNA in monkeySertoli cells. Addition of follicle-stimulating hormone (FSH)or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generatingagents and gonadotrophin-releasing hormone (GnRH) agonist orphorbol ester (PMA) to the cell culture significantly increasedtPA activity. PAI-1 activity in the culture was also enhancedby these reagents except 8-bromo-dibutyryl-cAMP, forskolin and3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPAactivity, whereas decreased PAI-1 activity, implying that neutralizationof PAI-1 activity by the high level of tPA in the conditionedmedia may occur. These data suggest that increased intracellularsignals which activate protein kinase A (PKA), or protein kinaseC (PKC) can modulate Sertoli cell tPA and PAI-1 activities.The concomitant induction of PA and PAI-1 by the same reagentsin the Sertoli cells may reflect a finely tuned regulatory mechanismin which PAI-1 could limit the excession of the proteolysis.     

Plasmin-catalysed cleavage of single chain tissue-type plasminogen activator in fibrin clots

The activity of single chain t-PA in the presence of fibrin was investigated and compared to that of its twochain counterpart. A plasmin resistant t-PA analogue in which Arg-275 is replaced by Gly was included in this study in order to avoid the complications caused by concomitant two chain generation during plasminogen activation experiments. Substantial plasminogen activation of single chain t-PA was observed during fibrin clot dissolution, still the clot lysis activity of two chain t-PA was found to be about 20% higher than that of the single chain form. Plasmin-catalysed cleavage of single chain t 25I-t-PA was studied in the presence and absence of fibrin. This reaction was not enhanced by fibrin, rather a small inhibition was observed. In addition to the primary cleavage site at Arg-275 Ile-276 secondary plasmin-catalysed cleavage resulting in a 30 000 Da fragment was observed. Appearance of this fragment took place roughly at the same time as a decline in clot lysis activity (but not in amidolytic activity) was observed. Secondary plasmin cleavage was not observed when fibrin was present.     

Meiotic and cytoplasmic maturation of oocytes collected in stimulated cycles is asynchronous

Oocytes collected in stimulated cycles are more readily fertilizedafter preincubation than are oocytes inseminated immediatelyafter collection. It has not been ascertained, however, whetherthis increase in the fertilization rate is due to extrusionof the first polar body (meiotic maturation) during this period,or to some conclusive cytoplasmic maturation subsequent to thepolar body extrusion. When analysing oocytes with a polar body(n = 14) by transmission electron microscopy, differences wereobserved in the appearance of cytoplasmic features which werecorrelated to the total durations both of systemic human chorionicgonadotrophin influence before collection and of oocyte culture.These differences were manifested as a delayed formation inaggregates of the smooth endoplamic reticulum in the ooplasmof oocytes having a polar body and might have signified a cytoplasmicmaturation. The degree of synchrony in the oocytes varied andthis could explain why some oocytes can be fertilized when inseminatedshortly after collection and others not until 8 h or even moreafter collection. Thus, although meiotic and cytoplasmic maturationis likely to be synchronized at ovulation of an oocyte in anatural cycle, they appear to be mainly asynchronous in oocytescollected in stimulated cycles.