Detection of vitronectin by ligand blotting with type 1 plasminogen activator inhibitor

Ligand blotting procedures were developed for the detection of type 1 plasminogen activator inhibitor (PAI-1) binding protein(s) (BPs) transferred to nitrocellulose sheets after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Purified vitronectin (Vn), a well characterised PAI-1 BP, was employed to optimise this assay system. After blocking with casein, the sheets were washed and then incubated with either ¹²⁵I-labelled PAI-1 (direct assay), or with unlabelled PAI-1 followed by a polyclonal antiserum to PAI-1 (indirect assay). In the latter case, the bound antibody was detected by using an ¹²⁵I-labelled second antibody. Binding was dose-dependent with respect to both Vn and PAI-1, and only active PAI-1 bound to Vn (i.e. latent PAI-1 and PAI-1 in complex with tissue-type plasminogen activator (t-PA), did not bind to Vn in this system). Moreover, t-PA, arginine and acidic conditions dissociated PAI-1 from Vn adsorbed to nitrocellulose. Analysis of bovine plasma by these techniques revealed the presence of a single PAI-I BP, and this protein was recognised by antisera to Vn. These results indicate that Vn previously fractionated by SDS-PAGE and transferred to nitrocellulose, continues to bind to PAI-I in a manner that resembles its behaviour in plasma and on extracellular matrix. These ligand blotting procedures may thus represent useful new approaches for the detection of other SDS-stable PAI-1 binding proteins in biological samples.     

Tissue plasminogen activator and plasminogen activator inhibitor type 1 gene polymorphism in patients with gastric ulcer complicated with bleeding

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) may be involved in the pathogenesis of peptic ulcers through suppression of fibrinolysis. This study was designed to investigate associations of t-PA and PAI-1 genes with clinical features of the patients with bleeding gastric ulcers. Eighty-four patients with peptic ulcers and 100 controls were studied between January 1998 and April 2000. We used polymerase chain reaction and endonuclease digestion to genotype for 4G/5G polymorphism in the promoter region of the PAI-1 gene and the Alurepeat insertion/deletion (I/D) polymorphism in intron h of the t-PA gene. Various clinical features, including lesion site, bleeding event, recurrence of ulcer, and rebleeding, were assessed using a multiple logistic regression model. The genotype distributions of both the t-PA and PAI-1 genes did not differ between the patient and control groups. The occurrence of the I/D or D/D genotype of t-PA was significantly higher in cases of duodenal ulcer (adjusted OR=4.39, 95% CI=1.12-17.21). When a dominant effect (i.e., 4G/4G or 4G/5G versus 5G/5G) of the 4G allele was assumed, the PAI-1 4G/4G genotype was independently associated with rebleeding after hemostasis (adjusted OR=5.07, 95% CI=1.03-24.87). Our data suggest that t-PA gene polymorphism is associated with duodenal ulcers, and that the PAI-1 gene may be a risk factor leading to recurrent bleeding after initial hemostasis.     

Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies

The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological and pathological extracellular tissue remodelling processes. uPA can also cleave uPAR, resulting in liberation of the amino-terminal domain I, which encompasses binding sites for both uPA and the adhesion molecule, vitronectin. In order to localise the different uPAR forms on the plasma membrane of murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III in uPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in the presence or absence of saturation with the amino-terminal fragment of uPA, ATF. However, the signal intensity obtained using another uPAR domain I specific mAb, mR1, was significantly reduced upon ATF saturation. Furthermore, when adding ATF, mR4 selectively stained the cleaved receptor. Applying these newly generated mAbs, we additionally demonstrated that cleaved and intact uPAR was evenly distributed on the surface of these cells.     

Diverse inhibition of plasminogen activator inhibitor type 1 by theaflavins of black tea

Fruits, vegetables, spices and a variety of teas are suggested for the prevention of many diseases. They encompass active, non-nutritional ingredients called nutraceuticals which are defined as food products that provide health benefits. Many nutraceuticals have been tested to identify inhibitors of plasminogen activator inhibitor (PAI-1). PAI-1 is the major and fast acting physiological inhibitor of fibrinolysis. However, preclinical studies of PAI-1 inhibitors have revealed an additional role of PAI-1 in the pathogenesis of vascular remodeling, renal injury, diabetes, obesity, Alzheimer's disease and cancer. Thus PAI-1 is a potential therapeutic target in some of these diseases. Our previous study revealed that a black tea extract (containing mostly theaflavins) inhibits PAI-1. In this study we report results for four pure (>98%) theaflavins. Inactivation of PAI-1 was tested by clot formation and by its lysis using thromboelastometry and measurements of human plasma turbidity. Among four tested theaflavins, theaflavin-3'-gallate was the most potent in PAI-1 inhibition trailed by theaflavin-3,3'-digallate, while the other two i.e., theaflavin and theaflavin-3-gallate did not show inhibitory activity.     

Characterization of a panel of monoclonal antibodies against human tissue-type plasminogen activator

Five murine monoclonal antibodies have been produced against the 1-chain form of human tissue plasminogen activator (t-PA). Affinity constants, calculated from data generated in a solid phase radioimmunoassay, ranged from 4.9 x 10(8)M-1 to 2.3 x 10(9) M-1 for these antibodies. This panel was classified into three groups based on the antibodies' effects on both the plasminogen activation and amidolytic activities of t-PA: complete inhibitors (CD2), partial inhibitors (DB10), and those with limited effects (AE5, BA10 and EG2). This same pattern for the five antibodies was observed in an assay measuring the binding of 125I-t-PA to its fast-acting inhibitor, PAI-1. It is concluded that this panel of antibodies defines at least three domains on the t-PA molecule, including its catalytic site.     

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.     

Low levels of plasminogen activator inhibitor type 1 in patients with inflammatory bowel disease

Recent investigations suggest that microthrombi formation in bowel capillaries could be a determinant factor in inflammatory bowel disease (IBD) pathogenesis. To evaluate the implication of fibrinolysis in these thrombotic events, we analysed plasmatic values of physiologic activators, effectors and inhibitors of fibrinolysis. In a sample of 112 IBD patients we found decreased plasminogen activator inhibitor type 1 (PAI-1:Ag) levels, a finding which has not been reported to date: 8.8±1.1 ng/mL (mean±SEM) versus 17.8±1.1 ng/mL in controls (p<0.0001). Urokinase-type plasminogen activator (u-PA:Ag) from patients with inflammatory activity was decreased: 0.41±0.03 ng/mL in active disease versus 0.52±0.02 ng/mL in inactive disease (p=0.01) and the same applied to patients with Crohn's disease (CD) (0.38±0.03 ng/mL) with respect to ulcerative colitis (UC) patients (0.52±0.025 ng/mL), p=0.001. Levels of plasminogen, alpha 2 antiplasmin and tissue-type plasminogen activator (t-PA:Ag) showed no differences with respect to the controls. With the exception of u-PA:Ag, there were no differences between UC and CD. These results demonstrate plasmatic disturbances in the flbrinolytic system of IBD patients.     

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     

Presence of a fast-acting specific inhibitor of plasminogen activator in human parotid saliva

The plasminogen activator in parotid saliva has recently been characterized as a tissue-type plasminogen activator (t-PA). In this study stimulated parotid saliva from 21 healthy volunteers was analysed for (a) t-PA activity using the fibrin plate assay or a bioimmunoassay coupled to the plasmin-specific chromogenic substrate S-2251, (b) t-PA antigen using an enzyme-linked immunospecific assay and (c) activity of the specific fast-acting plasminogen activator inhibitor (t-PA inhibitor) assayed by its inhibitory effect on one-chain t-PA added to the samples. In parotid saliva the median t-PA activity was 0.2 IU ml-1 (normal range 0.05-0.35 IU ml-1), but activity of free t-PA could not be demonstrated by bioimmunoassay in any sample. The mean antigen concentration of t-PA was 2.2 IU ml-1 in the 10 samples with levels above the detection limit (0.5 IU ml-1). High activity of t-PA inhibitor was demonstrated in all parotid salivas, and the mean inhibitory effect on t-PA was 13.2 IU t-PA quenched per millilitre. This study thus demonstrates a fibrinolytic system in parotid saliva characterized by high t-PA inhibitor activity and relatively low concentration of inactive, probably complex-bound, t-PA which is activated in the presence of fibrin.     

Tissue plasminogen activator and plasminogen activator inhibitor in patients with liver cirrhosis

The behaviour of tissue plasminogen activator (t-PA) and t-PA inhibitor (PAI) was studied in patients with decompensated liver cirrhosis. t-PA antigen showed a 3-fold significant increase with respect to healthy volunteers. t-PA activity in these patients did not significantly differ from that found in the controls, but the specific activity of t-PA was significantly lowered by 63%. PAI activity was significantly reduced in cirrhosis (−67%), while PAI-1 antigen was increased by 24%. Venous occlusion of the arm for 20min induced similar increases of t-PA antigen both in normal subjects and cirrhotic patients. These findings show that the total amount of t-PA is enhanced in liver cirrhosis, with no increase of t-PA activity due to the capacity of PAI to bind increased circulating t-PA antigen. The increased total amount of t-PA in cirrhotic patients could be explained in terms of a reduced clearance by the hepato-endothelial system.     

Tissue plasminogen activator and placental plasminogen activator inhibitor in human gingival fluid

Gingival crevicular fluid (GCF) is an extracellular exudate protecting periodontal tissue. Pathological changes in the periodontium are reflected in the composition of GCF. Crevicular fluid was collected from healthy volunteers on Millipore^® filter disks, and eluted at 100-fold dilution. The samples were tested for fibrinolytic activity and the presence of the plasminogen activators, t-PA and u-PA, and the specific plasminogen activator inhibitors, PAI-1 and PAI-2. In the diluted samples, t-PA was found at concentrations of 4–33 gmg/l, and PAI-2 at concentrations of 19–84 μg/l, whereas u-PA and PAI-1 were hardly detectable. Analyses of parotid and whole saliva yielded no evidence of gingival fluid contamination from these sources. The fibrinolytic activity of gingival fluid was completely quenched both by antibodies against t-PA and by PAI-2, indicating the presence of t-PA in its two chain form which is more susceptible to inhibition. This inhibition by PAI-2 may serve a regulatory purpose and prevent excessive proteolysis and tissue destruction.     

Effects of the Magenstrasse and Mill operation for obesity on plasma plasminogen activator inhibitor type 1, tissue plasminogen activator,fibrinogen and insulin

Plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (t-PA), fibrinogen and insulin were measured in 43 patients 3 years after they had undergone the Magenstrasse and Mill (MM) procedure and in 43 morbidly obese (MO) patients. Mean plasma PAI-1 was 61 ng/ml in the MO group compared to 30 ng/ml in the MM group (p < 0.0001); mean plasma t-PA was 10 ng/ml in the MO group compared to 7 ng/ml in the MM group (p < 0.001). Mean fibrinogen was 3.6 g/l in the MO group compared to 3.2 g/l in the MM group (p < 0.05). Mean plasma insulin levels were 32 U/ml in the MO group compared to 15 U/ml in the MM group. These changes suggest that use of the MM procedure may reduce mortality and morbidity from coronary heart disease in these high-risk obese patients.     

Diabetic nephropathy and plasminogen activator inhibitor 1 in urine samples

Plasminogen activator inhibitor-1 (PAI-1) may contribute to renal fibrosis because of its involvement in matrix (ECM) accumulation through inhibition of plasmin-dependent ECM degradation. The aim of this study is to determine urinary PAI-1 concentrations and its intrarenal localization in patients with various renal diseases and to identify inducers for PAI-1 expression in human cultured proximal renal tubular cells (HRCs). Urinary PAI-1 concentrations were significantly higher in patients with overt diabetic nephropathy (DN, n=36) than in proliferative glomerulonephritis (PGN, n=8), nephrotic syndrome (NS, n=10) and healthy controls (n=12). Urinary PAI-1 concentrations (ng/gCr) were directly correlated with urinary N-acetyl glucosaminidase (NAG) levels (r=0.58, p<0.05). As for intrarenal localization of PAI-1 antigen, strong stainings for PAI-1 were observed in proximal tubular cells of renal biopsy samples from patients with DN, while no stainings for PAI-1 were found in renal tissues of PGN or NS. Immunoblot analysis revealed the presence of PAI-1 protein in whole cell lyzates from HRCs grown to semiconfluency. Exposure of growth-arrested HRCs with hypoxia (1% O2) or TNF-alpha (10 ng/ml) for 24 hours increased the secretion rate of PAI-1 protein by about 2.0-fold, while 24-hour treatment with high glucose (450 mg/dl) did not increase PAI-1 secretion at all, compared with that of the control cells under normal glucose (100 mg/dl) and normoxia (18% O2). These findings suggest that PAI-1 expression is upregulated especially in the proximal renal tubular cells of DN, which may be explained partially by hypoxia and inflammatory cytokines but not high glucose.     

Highly stable plasminogen activator inhibitor type one (VLHL PAI-1) protects fibrin clots from tissue plasminogen activator-mediated fibrinolysis

Plasminogen activator inhibitor-1 (PAI-1) is the major specific inhibitor of tissue-type plasminogen activator (tPA) which mediates fibrin clot lysis through activation of plasminogen. Wild-type-PAI-1 (wPAI-1) is rapidly converted to the latent form (half-life of approximately 2 h) and loses its ability to inhibit tPA. We developed a very long half-life PAI-1 (VLHL PAI-1), a recombinant protein with a half-life >700 h compared with wPAI-1. In this study, VLHL PAI-1 was assessed for its ability to inhibit clot lysis in vitro. Clot formation was initiated in normal plasma supplemented with tPA by the addition of either tissue factor or human recombinant FVIIa. Clot lysis time, monitored turbidimetrically in a microtiter plate reader, was determined at various concentrations of wPAI-1 and VLHL PAI-1. Both wPAI-1 and VLHL PAI-1 caused a significant increase in clot lysis time, although the latter was somewhat less effective at lower concentrations. The VLHL PAI-1, but not wPAI-1, maintained its anti-fibrinolytic activity after preincubation overnight at 37 degrees. These studies demonstrate that VLHL PAI-1 is an effective inhibitor of fibrin clot degradation. Due to the high stability of VLHL PAI-1 compared with wPAI-1, this novel inhibitor of tPA-mediated fibrinolysis may have therapeutic applications for treating surgical and trauma patients when used directly or in conjunction with the procoagulant recombinant FVIIa.