Regulation of plasminogen activator inhibitor-1 secretion by urokinase and tissue plasminogen activator in rat epithelioid-type smooth muscle cells

Tissue plasminogen activator (tPA) and urokinase (uPA) are targets of plasminogen activator inhibitor-1 (PAI-1) inhibition. We have previously shown that both proteases can also induce PAI-1 secretion in rat smooth muscle cells (SMCs). We now report that both proteases appear to use very similar cellular mechanisms for signal transduction. They induced PAI-1 secretion using a pathway(s) involving protein kinase C (PKC). They also activated the Raf/Mek/mitogen-activated protein kinase (MAPK) pathway, which lies downstream of PKC activation. Activation of protein kinase A (PKA), however, lowered PAI-1 secretion induced by uPA and tPA, as a result of an inhibition of the PKC pathway and inhibition of Raf, Mek and MAPK phosphorylations. Src and syk family non-receptor tyrosine kinases (TK) were also involved in PAI-1 induction. The mechanisms of interaction of these tyrosine kinases with other pathways appeared to be quite different: src appeared to act within the PKC and PKA pathways, while syk operated independently of these pathways. Furthermore, whereas src inhibition resulted in inhibition of Raf/Mek/Erk phosphorylations, syk inhibition could only inhibit Mek and Erk phosphorylations but not the phosphorylation of Raf. These multiple pathways utilized by uPA and tPA to modulate PAI-1 secretion might be involved in determining the proteolytic or antiproteolytic potential of the SMCs under different pathophysiological conditions.     

Increased levels of tissue plasminogen activator with a low plasminogen activator inhibitor-1 in a patient with postoperative bleeding

Recurrent postoperative bleeding in a previously healthy man with a moderate prolongation of PT and PTT which did not correct with vigorous fresh-frozen plasma infusion was identified as a unique abnormality of the fibrinolytic system: a combination of an excess of tissue plasminogen activator (t-PA) at 300% of normal activity accompanied by a concurrent deficiency of plasminogen activator inhibitor-1 activity (PAI-1) of 10-20% of normal activity. This observation underscores the potentially important regulatory role of PAI-1 in the degree to which clinical manifestations of bleeding tendencies may occur in patients in whom an excess amount of t-PA is expressed.     

Subcutaneous abdominal, but not femoral fat expression of plasminogen activator inhibitor-1 (PAI-1) is related to plasma PAI-1 levels and insulin resistance and decreases after weight loss

Aims/hypothesis: Abdominal fat produces plasminogen activator inhibitor-1 (PAI-1) and could contribute to increased plasma PAI-1 values in human obesity associated with insulin resistance. Femoral fat, which is not associated with insulin resistance, is thought to be metabolically different from the abdominal fat. This study aimed to assess PAI-1 expression in these two fat territories in obese and lean subjects and to determine if concomitant changes of plasma and adipose tissue PAI-1 values occur after weight reduction. Methods: In 24 obese and 16 lean subjects, PAI-1 expression in abdominal and femoral subcutaneous fat, plasma PAI-1, insulin, triglyceride concentrations and insulin resistance were determined at the start of the study and in obese subjects after a 3-month weight reduction programme as well. Results: PAI-1 mRNA content in the abdominal subcutaneous fat was higher in obese than in lean subjects and positively correlated with plasma PAI-1 values (p < 0.01) and markers of insulin resistance (p < 0.05). In 18 obese subjects, re-examined after successful dieting, PAI-1 mRNA content decreased in the abdominal subcutaneous fat along with plasma PAI-1. However, the absolute changes of these two variables were not associated. In contrast, PAI-1 mRNA content in the femoral subcutaneous fat did not differ between lean and obese subjects, was not associated with plasma PAI-1 values or with markers of insulin resistance, and did not change after weight loss. Conclusion/interpretation: Only the abdominal, but not the femoral subcutaneous fat PAI-1 expression is a potential contributor to increases in plasma PAI-1 in obesity. Both plasma and abdominal subcutaneous fat PAI-1 values decreased significantly after weight reduction, although their absolute changes were not associated. [Diabetologia (2001) 44: 2025–2031] Received: 19 March 2001and in revised form: 3 August 2001     

The role of plasminogen activator inhibitor-1 as inhibitor of platelet and megakaryoblastic cell adhesion

In the present study the ability of plasminogen activator inhibitor type-1 (PAI-1) to interfere with platelet and megakaryoblastic cell adhesion was investigated. Both cell types exhibited integrin-dependent adhesion in a static system, mediated by alphaIIb beta3 on platelets and alpha v-integrins on different megakaryoblastic cell lines, even though they also expressed alphaIIb beta3. In a concentration-dependent manner, active, but not latent or complexed, PAI-1 abrogated cell adhesion onto vitronectin but not onto fibrinogen or other matrix substrata. Urokinase as well as thrombin neutralized the anti-adhesive effect of active PAI-1. The direct binding of vitronectin, but not of other matrix proteins, to integrin alphaIIb beta3 was blocked by active PAI-1 in a purified system. Since activated platelets release active and latent PAI-1 as well as structurally and functionally distinct forms of vitronectin, the described interactions appear to be physiologically significant. Co-distribution of vitronectin and PAI-1 at sites of fibrin polymers within platelet thrombi was demonstrated by transmission electron microscopy, suggesting an extracellular functional relationship of both release products with regard to cell adhesion. Our data emphasize the regulatory role of active PAI-1 in platelet adhesion to provisional matrix proteins as found during wound healing independent of its anti-proteolytic activity. Furthermore, megakaryocyte maturation may depend on the intact vitronectin-integrin adhesion system that is influenced by PAI-1, thereby proposing a regulatory role for the inhibitor in cellular differentiation.     

Expression of urokinase-type plasminogen activator receptor and plasminogen activator inhibitor-1 in gastric cancer

In gastric cancer, the urokinase-type plasminogen activator (uPA) system plays important roles in invasion and metastasis, processes which entail proteolysis and adhesion. Both the urokinasetype plasminogen activator receptor (uPAR) and the plasminogen activator inhibitor-1 (PAI-1) are thought to be important factors in this system. To clarify the relationship between these two factors and gastric cancer invasiveness, we evaluated the expression of uPAR and PAI-1 in 91 cases of gastric cancer by immunohistochemistry and in situ hybridization. Urokinase-type plasminogen activator receptor-mRNA, PAI-1-mRNA, uPAR and PAI-1 protein were diffusely distributed in the cytoplasm of the cancer cells and concentrated at invasive foci. Urokinase-type plasminogen activator receptor protein expression correlated with lymphatic, venous invasion (P<.01) and lymph node metastasis (P<0.05); uPAR-mRNA expression correlated with lymphatic, venous invasion and lymph node metastasis (P<0.05). Plasminogen activator inhibitor-1 protein expression correlated with lymphatic, venous invasion, lymph node metastasis and depth of invasion (P<0.01); PAI-1-mRNA expression was linked to lymphatic, venous invasion (P<0.01), lymph node metastasis and depth of invasion (P<0.05). This suggests that the proteolytic activity of uPAR and the cellular motility of PAI-1 in gastric cancer cells may determine penetration of lymphatic and blood vessels, whereby lymph node metastasis may be promoted and that the promotion of cellular motility by PAI-1 may influence the depth of cancer invasion.     

Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at M(r) of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of approximately 110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph.     

Immunohistochemical localization of plasminogen activator inhibitor-1 in human coronary atherosclerotic lesions involved in acute myocardial infarction

Summary Immunohistochemical localization of plasminogen activator inhibitor-1 (PAI-1) was studied using the streptavidin-biotin method in human atherosclerotic coronary arteries. The patients (one male and two females), whose ages ranged from 61 to 78 years, died of anteroseptal acute myocardial infarction without having received any thrombolytic therapy. PAI-1 immunoreactivities (IRs) were mainly detected in the endothelial cells, smooth muscle cells, and collagen fibers of the coronary arterial intima and not in thrombi. Remarkable immunohistochemical staining was seen in intimal collagen fibers. In one patient, intimal PAI-1 IRs partly bordered a thrombus and surrounded a large atheroma rich in cholesterol crystals. Our results suggest that PAI-1 is present in both cellular and extracellular components of human coronary atherosclerotic lesions.     

培哚普利对慢性心力衰竭患者血浆t-PA和PAI-1水平的影响

目的评价培哚普利对慢性心力衰竭(CHF)患者血浆组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制物-1(PAI-1)水平的影响。方法采用酶联免疫吸附法测定60例CHF患者(CHF组)及20例健康人(正常对照组)血浆t-PA、PAI-1水平。CHF组患者又随机均分为常规治疗亚组和培哚普利亚组。培哚普利亚组在常规治疗基础上加用培哚普利2~4mg,每日1次。所有CHF患者治疗2周后复测血浆t-PA、PAI-1水平。结果CHF患者血浆t-PA、PAI-1水平比正常对照组明显增高(P<0.01)。治疗后,培哚普利亚组血浆PAI-1水平比常规治疗亚组明显降低(P<0.01),血浆t-PA水平比常规治疗亚组明显升高(P<0.01)。结论培哚普利不仅可降低PAI-1水平,而且可升高t-PA水平,改善内源性纤溶功能。     

Impact of adipose tissue on plasma plasminogen activator inhibitor-1 in dieting obese women.

The increased incidence of cardiovascular diseases in obese subjects could be partially attributed to impaired fibrinolysis due to elevated plasma levels of tissue plasminogen activator inhibitor 1 (PAI-1). The associations between changes in plasma PAI-1, metabolic variables, and adipose tissue during weight loss and regain were studied in 52 healthy, premenopausal, obese women participating in a weight reduction program with a hypocaloric diet. PAI-1, insulin, triglyceride, leptin, and adipsin levels were determined at entry, after the first week, after completion of the program, and after 5 months of follow-up. In the 33 obese women who completed the program, decreases in PAI-1 antigen (-54%), PAI activity (-74%), and leptin (-51%), but not of adipsin, were observed. Changes in PAI-1 were associated with changes in body mass index (BMI), body fat, leptin, and insulin. The decreased level of PAI-1 remained low after follow-up in the 14 women who maintained their reduced weight but increased in the 16 women who regained weight. This increase in PAI-1 was correlated with an increase in body fat and leptin. On multivariate analysis, BMI was the major determinant of PAI-1 level. In conclusion, during weight reduction with a hypocaloric diet, the decrease in PAI-1 is more closely related to changes in adipose tissue than to changes in metabolic variables, suggesting a significant role for adipose tissue in regulating plasma levels of PAI-1.     

纤溶酶原激活物抑制剂-1与急性心肌梗死关系的研究

目的探讨纤溶酶原激活物抑制剂-1(PAI-1)在急性心肌梗死(AMI)发病中的作用。方法应用发色底物法测定中国汉族AMI患者56例(男44,女12,平均年龄67±10岁)和正常对照组55例(男42,女13,年龄68±8岁)血浆PAI-1活性,并分析环境因素对PAI-1水平的影响。结果 (1)AMI组和正常对照组的血浆PAI-1水平分别为O.90±O.13Au/ml和O.72±O.13Au/ml,两组间比较有显著性差异(P<0.001)。(2)吸烟、高血压病史、既往血栓性疾病病史、冠心病家族史、体重指数(BMI)、三酰甘油(TG)、血糖水平均与血浆PAI-1水平呈正相关,多元逐步回归分析显示,高血压病史、冠心病家族史是血浆PAI-1水平的独立预测因素。结论血浆PAI-1水平增加是心肌梗死(MI)的危险因素;(2)高血压、冠心病家族史为PAI-1水平的独立决定因素。     

Gene polymorphisms of tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with antiphospholipid antibodies

OBJECTIVE: Impaired fibrinolytical outcomes may be one of the pathogenic factors for thrombotic events in patients with antiphospholipid antibodies (aPL). We investigated the consequences of the gene polymorphisms of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) in patients positive for aPL. METHODS: Seventy-seven Japanese and 82 British patients with aPL were examined for Alu-repeat insertion (I)/deletion (D) polymorphism of the tPA gene by polymerase chain reaction (PCR), and 4G/5G polymorphism in the PAI-1 promoter gene by site-directed mutagenesis-PCR and restriction fragment length polymorphism analysis. Correlations between these polymorphisms and clinical symptoms of antiphospholipid syndrome (APS) (arterial thrombosis, venous thrombosis, miscarriage) were analyzed. RESULTS: Significant differences in the allele frequencies of these genes did not exist between patients and controls. There was no significant correlation between these gene polymorphisms and clinical symptoms of APS in patients with aPL. CONCLUSION: Polymorphisms of the tPA or PAI-1 genes probably do not significantly influence the risk of anerial thrombosis, venous thrombosis, or pregnancy morbidity in patients with aPL.     

Protection by α2-macroglobulin of tissue plasminogen activator against inhibition by plasminogen activator inhibitor-1

Tissue plasminogen activator (tPA) is widely used in the treatment of acute myocardial infarction (MI). However, its thrombolytic efficacy does not correlate with the dose administered. The interactions between tPA, α2-macroglobulin (α2-M), and plasminogen activator inhibitor-1 (PAI-1) were investigated both in vitro and in patients undergoing tPA therapy for MI in an attempt to identify variables that might affect the clinical efficacy of tPA.
Purified α2-M (5.4 mg/ml) protected 16.0% or 22.4% of tPA (12.5 IU/ml) activity from inhibition by PAI-1 at 4 or 8 IU/ml in vitro . Of nine patients treated with 5–20 mega IU of tPA for MI, the plasma activity of tPA remained increased for 15–30 min after the cessation of infusion in eight; the patient who failed to exhibit a persistent increase in tPA activity had a low plasma concentration of α2-M. Total tPA activity, derived from the area under the activity-versus-time curve (AUC), showed a significant inverse correlation with the ratio of the plasma PAI-1 activity to the plasma α2-M concentration. Total tPA activity did not correlate with plasma PAI-1 activity or plasma α2-M concentration alone. Results suggest that α2-M, by binding to tPA, protects the latter against inhibition by PAI-1.     

Fatty acid binding protein expression in different adipose tissue depots from lean and obese individuals

Aims/hypothesis: This study investigated the expression of adipose tissue fatty acid binding proteins (FABPs) in subcutaneous and visceral human adipose tissue depots from lean and obese individuals. Methods: Adipocyte lipid binding protein (ALBP) and keratinocyte lipid binding protein (KLBP) expression was quantified by western blot in subcutaneous and omental adipose tissue from 20 obese and 9 lean individuals. RNA expression was quantified by Northern blot in the obese subjects. Results: In the obese subjects, ALBP protein and RNA expression was higher in subcutaneous compared with omental adipose tissue (increases of 31 ± 14 % and 40 ± 13 % respectively, both p < 0.05), whereas in the lean group, KLBP protein levels were 32 ± 9 % lower in subcutaneous fat (p < 0.03). However, the ALBP/KLBP ratio was greater in subcutaneous compared to omental adipose tissue from both lean and obese subjects: increases of 187 ± 71 % (p = 0.01) and 52 ± 23 % (p = 0.17) respectively for the protein ratio, and 21 ± 6 % for RNA (p = 0.01, obese individuals). In lean subjects, insulin concentrations correlated positively with the ALBP/KLBP protein ratio in both depots (both p≤ 0.03). Conclusion/interpretation: There are regional differences in adipose tissue FABP expression, which could be influenced by obesity. However, the ALBP/KLBP ratio is greater in subcutaneous than visceral adipose tissue in lean as well as in obese subjects. Investigation of adipose tissue FABPs could further our understanding of the role of fatty acids in the insulin resistance syndrome. [Diabetologia (2001) 44: 1268–1273] Received: 1 February 2001 and in revised form: 25 June 2001     

Tissue plasminogen activator,plasminogen activator inhibitor-1 and von willebrand factor in rheumatoid arthritis

Summary Tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF), all of endothelial origin and active in the haemostasis, were analysed in 74 patients with rheumatoid arthritis. The concentrations were related to extra-articular disease and to the incidence of thromboembolic events (TE) registered in a 2-year follow-up period. Patients with extra-articular disease had a significant increase in PAI-1 activity and reduced tPA release in the venous occlusion test. von Willebrand factor, PAI-1 and also haptoglobin and triglycerides were significantly increased in the group of patients who later suffered from TE. In a multiple regression model, in which cholesterol, triglycerides and lipoprotein (a) showed significant association with TE, vWF had the strongest additive explanatory value. No distinct acute phase pattern of PAI-1 was found in any patient subgroup.     

Circadian fluctuations of tissue plasminogen activator antigen and plasminogen activator inhibitor-1 antigens in vasospastic angina.

To elucidate the circadian variation of fibrinolytic components in vasospastic angina, plasma levels of tissue plasminogen activator antigen (t-PA), free plasminogen activator inhibitor antigen (free PAI-1), t-PA/PAI-1 complex, and total PAI-1 were measured in venous plasma samples. Samples were taken every 6 hours (6:00 AM, noon, 6:00 PM, and midnight) for 24 hours in 14 patients with vasospastic angina, in 9 patients with exertional angina, and in 19 normal subjects. Twenty-four-hour Holter monitoring (Holter monitor, Del Mar Avionics, Irvine, Calif.) was also carried out in all subjects. All of the fibrinolytic components showed circadian variation, with a peak level at 6:00 AM in every study group except for the t-PA/PAI-1 complex in the group of patients with exertional angina. The values for all or the fibrinolytic components at each sampling time were higher in patients with coronary artery disease than in normal subjects. In particular, the mean value of free PAI-1 at 6:00 AM in patients with vasospastic angina was significantly higher than that in normal subjects and that in patients with exertional angina. This value of free PAI-1 in patients with vasospastic angina was closely associated with the duration of ischemic attacks. These results suggested that the circadian fluctuation of fibrinolytic components may be an important factor that leads to coronary thrombosis at the time of coronary spasm, especially in the early morning.     

Plasminogen activator inhibitor-1 removal using dextran sulphate columns. Evidence of PAI-1 homeostasis

Patients with high plasma plasminogen activator inhibitor-1 (PAI-1) antigen levels are prone to develop thrombosis. Lowering PAI-1 levels may offer a therapeutic option and help to better understand PAI-1 metabolism. We examined the effect on plasma PAI-1 levels of LDL-apheresis using dextran sulphate (DS) columns in 12 patients (9 male, 3 female, 49 ± 10 years) with heterozygous familial hypercholesterolaemia and coronary artery disease. One plasma volume equivalent (2.3–4.0 l) was treated during each procedure (at flow rates of 23 ± 2 ml/min). Lipids and PAI-1 antigen levels were measured in plasma before and immediately after 19 aphereses (once in 7 patients, twice in 3 patients and three times in 2 patients) and also at 3 and 7 days post apheresis in five of these patients and in the column eluates from 8 of these patients. DS-apheresis reduced plasma cholesterol (50 ± 8%), triglyceride (45 ± 27%), apolipoprotein B (59 ± 10%) and PAI-1 antigen levels from 10.2 ± 5.2 to 6.0 ± 3.1 ng/ml (P = 0.005). The PAI-I changes were independent of circadian variation. PAI-I bound to the DS-columns (3.51 ± 1.03 ng/ml filtered plasma) and the percent of filtered PAI-1 that was bound correlated inversely (r = −0.81, P < 0.02) with basal PAI-1 levels indicating a high affinity saturable binding process. In four patients, plasma PAI-1 levels post-apheresis were higher than expected based on the amount of PAI-removed by the DS columns. The difference between the expected and actual PAI-1 level post apheresis, reflecting PAI-1 secretion or extracellular redistribution, correlated inversely with basal PAI-1 levels (r = −0.83, P = 0.01). PAI-1 levels returned to baseline pre-apheresis values 7 days post apheresis. PAI-1 antigen may be removed from plasma without adverse effect, resulting temporarily in its extracellular redistribution and restoration to baseline levels over one week. PAI-1 redistribution particularly when baseline pre-apheresis values were low may reflect a homeostatic mechanism to maintain sufficient PAI-1 levels. Procedures that could selectively remove PAI-1 from plasma may offer a treatment option for those with very high plasma PAI-1 levels and thrombosis.     

Synthesis and secretion of plasminogen activator inhibitor-1 by human preadipocytes.

To further investigate the role of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue physiology, the production and regulation of PAI-1 was determined in primary cultures of human preadipocytes. When expressed as production per cell and cultured under identical conditions, human preadipocytes from both visceral (omental) and sc depots of lean and obese individuals released significant, yet similar, amounts of PAI-1 protein into the conditioned medium. High steady-state PAI-1 messenger RNA (mRNA) concentrations were observed in visceral and sc preadipocytes, with the relative level of expression equivalent to beta-actin mRNA. Tumor necrosis factor alpha significantly decreased PAI-1 production in a concentration-dependent manner in both visceral and sc cultures, whereas transforming growth factor beta significantly elevated PAI-1 production, but only in sc preadipocytes from obese individuals. Addition of insulin had no effect on antigen levels in conditioned medium of preadipocyte cultures. Stimulation of the preadipocyte cultures with a defined medium resulted in differentiation to the adipocyte phenotype, as determined by flow cytometric analysis, verifying the cultures as human preadipocyte. These studies are the first to observe significant PAI-1 mRNA expression and protein production in primary cultures of a human adipose tissue cellular component, and they suggest that nascent adipocytes contribute significantly to the elevated plasma PAI-1 observed in obesity.