Low Plasminogen Activator Inhibitor Type 1 Research Articles

Tissue Plasminogen Activator as a Possible Indicator of Breast Cancer Relapse: A Preliminary, Prospective Study.

Simple SummaryBreast cancer is the most common malignant neoplasm and the leading cause of cancer death in women worldwide. The heterogeneity of breast cancer is a significant challenge facing modern medicine, especially at the stage of diagnosis. In recent years, a substantial role of fibrinolytic biomarkers in the pathogenesis of breast cancer has been proved as the primary cause capable of initiating local recurrence and metastasis to other organs. Due to its proteolytic properties, fibrinolytic protein activity may lead to subendothelial layer degradation and promote neoplastic cell motility. Thus, we examined the fibrinolytic profile markers’ prognostic value in predicting the disease’s relapse. Our studies indicate that the t-PA antigen and PAI-1 activity are biomarkers with high prognostic potential. We showed that a lower baseline plasma concentration of t-PA antigen and higher PAI-1 activity might be strong predictors for distant metastases and independent prognostic markers in breast cancer patients.(1) Background: The fundamental causes of breast cancer mortality are the cancer spread and hypercoagulability state. The study aimed to evaluate the prognostic efficacy of the fibrinolytic profile concerning 5-year follow-up. (2) Methods: We investigated the predictive potential of the plasma activity of urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 (PAI-1) as well as antigen of tissue plasminogen activator (t-PA), u-PA, PAI-1, and PAI-1/t-PA and PAI-1/u-PA complexes in 41 breast cancer patients. The median follow-up was 66 months, with full evidence of the first event. (3) Results: A significantly lower level of PAI-1 antigen was noted in IBrC patients with lymph node involvement (N1) than in patients with free lymph node metastases (N0). According to ROC curve analysis, a t-PA antigen was the strongest predictor of disease relapse (the area under the curve, AUC = 0.799; p < 0.0006). Patients with PAI-1 activity < 3.04 U/mL had significantly better disease-free survival (DFS) compared to those with PAI-1 activity > 3.04 U/mL. Patients with both t-PA antigen lower than 1.41 ng/mL (cut-off according to median value) and lower than 1.37 ng/mL (cut-off according to ROC curve) had significantly shorter DFS (p = 0.0086; p = 0.0029). (4) Conclusions: The results suggest that a higher plasma t-PA antigen level or lower PAI-1 activity are linked to better outcomes in breast cancer patients.

Evaluation of the prognostic value of fibrinolytic elements in invasive breast carcinoma patients.

Breast cancer (BrC) is one of the most serious oncological problems in the world. The aim of the study was to evaluate concentrations of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) and their ratios: t-PA/PAI-1 and PAI-1/t-PA in breast cancer patients and in healthy individuals and to estimate the ability of fibrinolytic parameters in predicting neoplasm disease and disease relapse. One hundred and five women were enrolled in the study, including 60 cases with primary BrC, (M0) and 45 healthy females. Follow-up was completed in all BrC patients with a 16.7% recurrence rate. An immunoassay of t-PA, PAI-1 in all cases was made as well as the immunohistochemistry of estrogen and progesterone receptors, human epidermal growth factor receptor 2, E-cadherin, and Ki-67 was performed in BrC subjects. A significantly higher PAI-1 concentration in breast cancer patients below the age of 55 than in controls was obtained. According to the ROC curve analysis, the PAI-1 concentration demonstrates the most accurate prognostic value with the cut-off point at 33.91 ng/ml, with 90% sensitivity and 36% specificity, which discriminates between controls and cancer patients. However, t-PA presents the highest area under the receiver-operating characteristic curves (AUCROC)=0.634 in predicting disease relapse with the cut-off value of 5.3 ng/ml. According to the Kaplan-Meier curves, a high concentration of t-PA (>5 ng/ml) and a lower PAI-1/t-PA ratio (<7.5) are associated with shorter survival. Evaluation of plasma t-PA and PAI-1 concentrations may deliver relevant prognostic information for breast cancer patients.

Plasminogen Activator Inhibitor Type 1 in Blood at Onset of Chemotherapy Unfavorably Affects Survival in Primary Ovarian Cancer.

Plasminogen activator inhibitor type 1 (PAI-1) belongs to the family of the plasminogen activator system. PAI-1 stimulates fibrinolysis and also promotes tumor progression. The aim of this study was to evaluate the prognostic value of blood plasma PAI-1 content in patients with epithelial ovarian cancer who start the first-line chemotherapy. PAI-1 content was measured in the blood of 61 patients with epithelial ovarian cancer at onset of first-line chemotherapy. The patients were further stratified into the low PAI-1 group (≤20ng/mL; 33 patients) and the high PAI-1 group (>20ng/mL; 28 patients). We found that the greater plasma PAI-1 content was associated with a significantly lower probability of a 5-year-long survival compared to that when PAI-I content was lower (45.5% vs. 69.5%, respectively; p=0.04). However, the risk of cancer recurrence within 5years failed to differ appreciably. A multivariate analysis revealed that the lower PAI-1 plasma content was an independent factor of longer overall survival (death risk ratio of 0.36, 95%CI=0.16-0.79; p<0.01). We conclude that PAI-1 is yet another biomarker of survival in patients with ovarian cancer.

Increased incorporation of antiplasmin into the fibrin network in patients with type 1 diabetes.

Diabetes is associated with various vascular complications and is suggested to induce a prothrombotic state. In the current study, we characterized antiplasmin incorporation into fibrin in relation to other fibrinolytic compounds in patients with type 1 diabetes. A total of 236 patients with type 1 diabetes and 78 control subjects were investigated. The incorporation of antiplasmin into the fibrin network and the plasma levels of plasminogen activator inhibitor type 1 (PAI-1) activity, tissue plasminogen activator (tPA) activity, tPA/PAI-1 complex, plasmin-antiplasmin complex, antiplasmin, factor XIII, and d-dimer were measured. In addition, we used global assays to study fibrinolysis. The incorporation of antiplasmin into the fibrin network was significantly higher in patients with type 1 diabetes than in control subjects without diabetes (1.65 ± 0.25 vs. 1.35 ± 0.18 mg/L, respectively; P < 0.0001). The patients also had lower PAI-1 activity (2.19 units/mL [interquartile range 0.96-5.42] vs. 4.25 units/mL [1.95-9.0]; P = 0.0012) and antiplasmin level in plasma (78.5 ± 13.3 vs. 83.2 ± 15.4 mg/L; P < 0.05), resulting in a higher fibrinolytic capacity (shorter clot lysis time; P = 0.0090). We did not find any important sex differences regarding fibrinolysis in the patients or in the control subjects. Patients with type 1 diabetes incorporate more antiplasmin into the fibrin network than control subjects without diabetes do and have a reduced PAI-1 activity and a shorter clot lysis time. These results suggest that patients with type 1 diabetes produce a fibrin clot that is more resistant to fibrinolysis, which, however, may be counteracted by an increased fibrinolytic potential in plasma.

Plasminogen activator inhibitor type 1 serum levels and 4G/5G gene polymorphism in morbidly obese Hispanic patients with non-alcoholic fatty liver disease

Background. The plasminogen activator inhibitor type-1 (PAI-1) has been implicated in the regulation of fibrinolysis and extracellular matrix components. The single base pair guanine insertion/deletion polymorphism (4G/5G) within the promoter region of the PAI-1 gene influences PAI-1 synthesis and may modulate hepatic fibrogenesis.Aim. To evaluate the influence of PAI-1 serum levels and 4G/5G polymorphism on the risk of liver fibrosis associated to non-alcoholic fatty liver disease (NAFLD) in morbidly obese patients.Material and methods. Case-control study of 50 obese patients undergoing bariatric surgery and 71 non-obese subjects matched by age and sex. Anthropometric and biochemical measurements were performed, including PAI-1 serum levels. Genomic DNA was obtained to assess the presence of 4G/5G polymorphism.Results. BMI, insulinemia, triglycerides, HOMA-IR, hypertension and diabetes were significantly higher in obese patients compared to control subjects. PAI-1 serum levels observed in obese patients were significantly lower (10.63 ± 4.82) compared to controls (14.26 ± 11.4; p < 0.05). No differences were observed in the PAI-1 4G/5G promoter genotypes frequencies (p = 0.12). No differences were observed in PAI-1 plasma levels among obese patients with liver fibrosis (10.64 ± 4.35) compared to patients without liver fibrosis (10.61 ± 5.2; p = 0.985). PAI-1 4G/5G promoter genotypes frequencies were similar in patients with or without liver fibrosis associated to NASH (p = 0.6).Conclusions. Morbidly obese patients had significantly lower PAI-1 serum levels with similar PAI-1 4G/5G genotypes frequencies compared to non-obese subjects. The frequency of 4G/5G genotypes in Chilean Hispanic healthy subjects was similar to that described in other populations. No association was found between PAI-1 serum levels or 4G/5G genotype with liver fi-brosis in obese patients.

Plasminogen activator inhibitor-1 C/G polymorphism in relation to plasma levels in rheumatoid arthritis

Plasminogen activator inhibitor type 1 (PAI-1) is an inhibitor of plasmin production. Plasmin can directly or indirectly to degrade cartilage and bone matrix. The PAI-1 HindIII polymorphism has been associated with high PAI-1 plasma levels in myocardial infarction patients and control populations. Furthermore, it has been associated with the angiographic extent of coronary artery disease, but their involvement in other diseases is still uncertain. Here, we assessed the relationship between PAI-1 HindIII polymorphism and PAI-1 plasma levels in rheumatoid arthritis (RA). One hundred and twenty-five RA patients and 132 control subjects (CS) were included. Genotypes were identified by the polymerase chain reaction-restriction fragment length polymorphism technique and PAI-1 plasma levels were quantified using an ELISA kit. Not significant differences in genotype and allele frequencies between both studied groups were observed (P > 0.05). RA patients showed lower PAI-1 plasma levels (18.92 +/- 12.94 ng/ml) than CS (23.68 +/- 23.38 ng/ml), without significant difference (P = 0.299). However, in RA patients the C/G genotype carriers showed higher PAI-1 plasma levels (23.00 +/- 13.81 ng/ml) with respect to C/C (16.77 +/- 11.97 ng/ml) and G/G (10.47 +/- 7.07 ng/ml) genotype carriers (P = 0.036). The PAI-1 HindIII polymorphism was not associated with RA susceptibility. However, the C/G genotype is associated with high PAI-1 plasma levels in RA patients.

Low PAI‐1 activity in relation to inflammatory parameters, insulin profile and body mass index

High plasminogen activator inhibitor type 1 (PAI-1) activity is associated with inflammatory reactions and insulin resistance, but it is unclear what regulates PAI-1 activity at the low end. The purpose of this study was to investigate if patients with low PAI-1 activity have a lack of inflammatory response or a low insulin level. Retrospective cohort study with internal controls. Sixty-three patients referred for investigation of bleeding tendency and with low PAI-1 activity were compared with 118 patients with normal or high PAI-1 activity. Levels of C-peptide, proinsulin, high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6). Adjustments were made for body mass index (BMI), oral oestrogens and age. Low PAI-1 activity was defined as less than 1 U mL(-1). Body mass index in the low normal range, oral oestrogens, young age and low C-peptide were significantly associated with low PAI-1 activity and there was a trend for association with IL-6 in univariable analysis. The effect of age disappeared after correction for oral oestrogens and the effect of C-peptide and IL-6 disappeared after further adjustments. Low BMI remained as the strongest predictor of low PAI-1 activity. Patients with bleeding tendency and low PAI-1 activity have inflammatory and insulin profiles similar to those with normal or high PAI-1, whereas BMI seems to be the most important determinant.

Relationship between maternal and cord blood hemostatic disturbances in preeclamptic pregnancies

Endothelial cell activation or damage is believed to play a key role in preeclampsia (PE) and may underlie the hemostatic changes observed in this syndrome. The aim of this study was to evaluate a relationship between maternal and cord blood hemostatic disturbances in preeclamptic pregnancies. We measured the plasma levels of tissue plasminogen activator (tPA) antigen and of plasminogen activator inhibitor type 1 (PAI-1) antigen, both markers of hemostatic and endothelial function, and fibrin fragment D-dimer. Maternal blood from uncomplicated ( n = 42) and PEc pregnancies ( n = 44) were collected before delivery, and umbilical cord blood (UCB) immediately after delivery. In preeclamptic cases, UCB presented significantly higher tPA values and significantly lower PAI-1/tPA ratio. Preeclamptic women also presented significantly higher tPA, as well as PAI-1 values, when compared with normal pregnant women; no significant difference was found for D-dimer. In preeclamptic women, proteinuria (a marker of PE severity) correlated positively and significantly with tPA and PAI-1 antigen levels. An inverse relationship between maternal tPA antigen levels and fetal birth weigh in PE was also observed. Our data show that the hemostatic maternal disturbances observed in preeclamptic women have similarities with the UCB circulation, and that endothelial dysfunction is the most plausible underlying cause. Moreover, maternal hemostatic disturbances seem to be associated with the severity of PE. Further studies are needed to strength the values of tPA and PAI-1 as markers of severity in PE.

Laboratory evidence of hyperfibrinolysis in association with low plasminogen activator inhibitor type 1 activity

Low activity of plasminogen activator inhibitor type 1 (PAI-1) has been associated with bleeding complications in surgery. We earlier reported a higher prevalence of low PAI-1 activity among patients with bleeding tendency as compared with normal control individuals. The present study evaluated whether low PAI-1 activity actually is associated with markers of increased fibrinolytic activity in plasma from patients with a history of bleeding. PAI-1 activity, plasmin-antiplasmin complex (PAP) and D-dimer were analyzed in plasma samples from 424 consecutive patients referred to the Coagulation Unit for investigation of bleeding symptoms. The median PAI-1 activity was 4.0 U/ml [interquartile range (IQR), 1-10 U/ml], the median PAP level was 1.59 mg/l (IQR, 1.40-1.91 mg/l) and the median D-dimer level was 71 microg/l (IQR, 46-111 microg/l). The median PAP concentration for patients with PAI-1 less than 1.0 U/ml was 1.73 mg/l (IQR, 1.53-2.30 mg/l), and that for PAI-1 of at least 1.0 U/ml was 1.54 mg/l (IQR, 1.36-1.83 mg/l) (P < 0.0001). There was also a significant difference between the PAP levels in patients with normal PAI-1 (1-15 U/ml) versus elevated PAI-1 (> 15 U/ml) (P = 0.024). The level of D-dimer did not correlate with PAI-1 activity. In conclusion, the activation of plasminogen measured as PAP was higher in patients with bleeding symptoms in combination with PAI-1 activity less than 1.0 U/ml than in those with PAI-1 activity of at least 1.0 U/ml. The coagulation activity under normal conditions, as measured by D-dimer, did not differ between the two patient subsets. The results support our previous definition of low PAI-1 as activity below 1.0 U/ml.

Plasminogen activator inhibitor type‐1 (PAI‐1) polymorphism 4G/5G is associated with prostate cancer among men with a positive family history

Variation in the expression of plasminogen activator inhibitor type-1 (PAI-1) is associated with many human diseases, including several types of cancer. In particular, tumor cell overexpression of PAI-1 has been found to inhibit prostate cancer tumor growth, angiogenesis, and metastasis in mouse models. Normal host cell expression of PAI-1 is influenced by the 4G/5G insertion/deletion polymorphism in the promoter region of the PAI-1 gene. To evaluate the effect of PAI-1 expression on cancer development, we examined the association of the 4G/5G polymorphism in a sibling-based case-control study of prostate cancer. One thousand one hundred thirty seven subjects, 655 cases, and 482 sibling controls from 526 families, were recruited from the major medical institutions in the greater Cleveland, OH area and from the Henry Ford Health System, Detroit, MI. A Cox age-of-onset model with robust variance estimation was used to evaluate the association between the PAI-1 4G/5G polymorphism and prostate cancer. No association was observed between the PAI-1 4G/5G polymorphism and prostate cancer in the entire sample. We did, however, identify a statistically significant association between the PAI-1 4G/5G polymorphism and prostate cancer in subjects with a family history of this disease (OR = 1.28, 95% CI 1.02-1.61, P-value = 0.036). The PAI-1 5G/5G genotype, associated with lower PAI-1 expression, appears to drive this result as it was associated with an increased risk of prostate cancer and an earlier mean age of onset compared to those with the 4G/4G genotype (OR = 1.83, 95% CI 1.12-2.99) while the 4G/5G genotype group did not show a significant difference in prostate cancer risk compared to the 4G/4G genotype group (OR = 0.98, 95% CI 0.75-1.28). These observations suggest that the 4G/5G polymorphism in PAI-1 may explain some of the increased risk and earlier mean age of onset of prostate cancer due to a positive family history.

Low PAI-1 activity in relation to the risk for perioperative bleeding complications in transurethral resection of the prostate

Introduction Low levels of plasminogen activator inhibitor type 1 (PAI-1) have been associated with increased risk for perioperative bleeding in some case reports. The aim of this study was to investigate prospectively whether low PAI-1 activity increases the risk for perioperative bleeding in patients undergoing transurethral resection of prostate, an organ with high fibrinolytic activity. Patients and methods 62 patients with benign prostatic hyperplasia planned for transurethral resection were included. Blood samples for PAI-1 were taken together with other routine preoperative blood samples 1 week before surgery but analyzed after the hospitalization. The intraoperative blood loss was determined by measuring the amount of hemoglobin in the irrigating fluid. The postoperative blood loss was estimated from calculations of hemoglobin mass (Hb mass), which is a product of hemoglobin concentration and blood volume. Hb mass was calculated before surgery and on the day of discharge, and was adjusted for intraoperative blood loss and transfused Hb mass. Bleeding complications were defined as re-operation due to bleeding, more than 40 ml intraoperative bleeding/g resected prostatic tissue or postoperative blood loss corresponding to more than 100 g of hemoglobin. Results Bleeding complications were observed in 3 of 4 (75%) patients with low PAI-1 levels, defined as < 1 U/ml, and in 16 of 58 (28%) patients with PAI-1 levels > 1 U/ml ( P = 0.082). After adjustment for resection time, resected prostatic mass and systolic blood pressure this became borderline significant (odds ratio 11.8; 95% confidence interval 1.00–139; P = 0.05). Conclusion Low PAI-1 activity may contribute to the risk of bleeding after transurethral resection of the prostate.

Evaluation of low PAI-1 activity as a risk factor for hemorrhagic diathesis.

Prospective studies of the epidemiology and clinical significance of low plasminogen activator inhibitor type 1 (PAI-1) activity are lacking. To evaluate the prevalence of low PAI-1 activity in patients with a bleeding tendency in comparison with a normal population. In 586 consecutive patients, referred because of bleeding symptoms, we added analyses of PAI-1 activity and tissue plasminogen activator complex with PAI-1 (t-PA-PAI-1) to the routine investigation, consisting of platelet count, bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII, von Willebrand factor activity, and antigen. Controls were 100 blood donors and 100 age- and sex-matched healthy individuals. The latter were also evaluated regarding the previous bleeding episodes. The bleeding history was classified as clinically significant or not, and the criteria were fulfilled in 75% of the patients and 18% of the healthy controls. The routine laboratory investigation of the patients was negative in 57%. Low PAI-1 activity, defined as <1.0 U mL(-1), was found in 23% of the patients and in 13% and 10% of the blood donors and healthy controls, respectively (odds ratio and 95% CI, 2.04; 1.11-3.77 and 2.75; 1.39-5.42, respectively). The difference remained statistically significant after the adjustment for body mass index, use of estrogens, sex and age (odds ratio for patients vs. healthy controls 3.23; 95% CI, 1.22-8.56, P = 0.019). The distribution of the 4G/5G genotypes in the patients was not different from that of two control populations. No specific symptom predicted for low PAI-1, which did not aggravate the clinical picture in association with the other hemostatic defects. Low tPA-PAI-1 was not associated with the increased bleeding tendency. Low PAI-1 activity is common in patients with a bleeding diathesis, but it is a risk factor of minor clinical importance and not associated with specific bleeding manifestations.

Increased plasma fibrinolytic activity in bleeding gastrointestinal angiodysplasia

Gastrointestinal angiodysplasia is a major cause of recurrent bleeding. Haemostatic abnormalities have been implicated in the haemorrhage from these common vascular lesions but their precise contribution remains to be established. Our aim was to investigate whether bleeding angiodysplasia is associated with any specific coagulation disorder. Clinical features and blood samples were prospectively obtained from 21 patients with bleeding gastrointestinal angiodysplasia 3 months after the last episode of haemorrhage. Plasma levels of von Willebrand factor, D-dimer, plasminogen activator inhibitor type 1 (PAI-1), tissue-plasminogen activator activity, tissue factor pathway inhibitor and activated factor VII (FVIIa-rTF) were measured. A group of 14 patients with bleeding duodenal ulcer were similarly studied as controls. Mean plasma von Willebrand factor levels were higher in angiodysplasia patients (208+/-12%) than in controls (143+/-11%) (P<0.05). D-dimer levels (661+/-80 ng/ml) and tissue-plasminogen activator activity levels (2.04+/-0.14 IU/ml) were also higher than in controls: 395+/-99 ng/ml and 1.6+/-0.1 IU/ml, respectively (P<0.05), whereas levels of PAI-1, FVIIa-rTF and tissue factor pathway inhibitor were similar in both groups. However, PAI-1 levels (31.5+/-11 ng/ml) were lower in high-bleeding-rate angiodysplasia (more than two bleeding episodes/year) than in low-bleeding-rate angiodysplasia (< or = 2 bleeding episodes/year) (PAI-1 47+/-14 ng/ml) (P<0.05). In a multivariate regression analysis, the plasma level of PAI-1 was a predictor of haemorrhage from angiodysplasia (P<0.05). Increased plasma fibrinolytic activity may contribute to bleeding from angiodysplasia. Low plasma PAI-1 levels constitute a risk factor for bleeding tendency in patients with angiodysplasia.

Upregulation of PAI-1 is mediated through TGF-β/Smad pathway in transplant arteriopathy

Background Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiologic inhibitor of plasminogen activator in vivo. Increased PAI-1 expression is associated with arteriosclerosis. Transforming growth factor-beta (TGF-β) induces PAI-1 production via Smads. Methods In vivo, TGF-β receptors (TβRs), Smad2, Smad3, and Smad4, PAI-1, and Smad2 phosphorylation were examined by immunohistochemistry in 3 native aortas, 14 rat aortic syngrafts, and 19 allografts collected at 15, 30, and 45 days post-transplantation. In vitro, phosphorylation of Smad2 and induction of PAI-1 mRNA in human aortic smooth muscle cells (SMCs) in response to TGF-β treatment were detected by Western blot and by TaqMan real-time RT-PCR, respectively. Results Immunohistochemical staining revealed that vascular parenchymal cells contained TβRI, TβRII, Smad2, Smad3, and Smad4, known signaling transducers for TGF-β/Smad pathway, in all samples. Intense staining for phospho-Smad2 was observed in 94% of endothelial cells (ECs), 86% of intimal cells, 27% of medial SMCs, and 38% of adventitial cells at all 3 time points in all aortic allografts, but only in 5% of ECs in syngrafts. PAI-1 immunoreactivity was detected in similar number of cells, and from consecutive sections, phospho-Smad2 colocalized with PAI-1, in the aortic allografts. Low basal level PAI-1 expression was observed in aortic syngrafts and native vessels. Smad2 phosphorylation and time-dependent PAI-1 induction were detected in cultured SMCs upon TGF-β treatment. Conclusions Phospho-Smad2 staining in aortic allografts indicates the activation of TGF-β signaling in allo-transplantation; and co-localization of PAI-1 and phospho-Smad2 suggests that PAI-1 upregulation is mediated mainly by TGF-β/Smad pathway in aortic allografts.

PAI-1 and EGFR expression in adult glioma tumors: toward a molecular prognostic classification

Purpose : Molecular classification of gliomas is a major challenge in the effort to improve therapeutic decisions. The plasminogen activator system, including plasminogen activator inhibitor type 1 (PAI-1), plays a key role in tumor invasion and neoangiogenesis. Epidermal growth factor receptor (EGFR) is involved in the control of proliferation. The contribution of PAI-1 and EGFR to the survival of gliomas was retrospectively investigated. Methods and Materials : Fifty-nine adult gliomas treated by neurosurgery and conventional irradiation were analyzed, including 9 low-grade (2) and 50 high-grade (3–4) tumors (WHO classification). PAI-1 was measured on cytosols and EGFR on solubilized membranes using ELISA methods. Results : High PAI-1 levels were strongly associated with high histologic grade ( p < 0.001) and histologic necrosis ( p < 0.001). PAI-1 also correlated positively with patient age ( p = 0.05) and negatively with Karnofsky index ( p = 0.01). By univariate analysis of the high-grade population, higher PAI-1 ( p < 0.0001) and EGFR values ( p = 0.02) were associated with shorter overall survival. Only PAI-1 was an independent factor in multivariate analysis. Grade 3 tumors with low PAI-1 (100% 3-year overall survival rate) presented the same clinical outcome as the low-grade tumors. Conclusions : In this prognostic study, PAI-1 and EGFR expression revealed similarities and differences between high-grade gliomas that were not apparent by traditional clinical criteria. These data strongly support that biologic factors should be included in glioma classification and the design of clinical trials to treat more homogeneous populations.

Hemostatic variables as independent predictors for fetal growth retardation in preeclampsia

Preeclampsia is a major contributor to perinatal disease and fetal growth retardation (FGR). It has been suggested that increased intravascular coagulation, fibrin deposition in spiral arteries and hypoperfusion of the placenta are involved in these pregnancy complications. Multiple variables of the hemostatic system and lipid metabolism, as well as clinical features, were entered into univariate and multivariate models in order to examine the association with preeclampsia and FGR. Two hundred women with preeclampsia and 97 normotensive pregnant women were examined. Plasma levels of the thrombin-antithrombin complex (TAT), tissue factor pathway inhibitor free antigen (TFPI-Fag), protein S free antigen, plasminogen activator inhibitor type-1 (PAI-1) activity and serum levels of triglycerides were significantly increased, whereas plasma levels of antithrombin (AT), fibrinogen, C4b-binding protein (C4b-BP), PAI-2 antigen and serum HDL-cholesterol levels were decreased in the presence of preeclampsia. In the multivariate regression analysis, high TFPI-Fag plasma levels were associated with the presence of preeclampsia. The presence of FGR was in the univariate analysis associated with decreased PAI-1 activity and lower concentrations of fibrin, fibrinogen, factor VII antigen and PAI-2 antigen, as well as with evidence of macroscopic placental infarction. In a multivariate regression model, low maternal weight, placental infarction and low PAI-2 levels were predictors for low birth weight. In a logistic regression model, with the presence or absence of FGR as the dependent variable, male sex of the infant, placental infarction, low PAI-1 activity and factor VII antigen or PAI-2 antigen levels were independent predictors. Our results are consistent with activated coagulation in the placental vessels in preeclampsia. A low concentration of PAI-2 antigen in plasma emerged as the most consistent risk factor for preeclampsia and FGR.

Fibrinolytic Activity in Peripheral Atherosclerosis in the Elderly

Increased concentrations of plasminogen activator inhibitor type 1 (PAI-1) and of D-dimer have jointly been found in subjects with cardiovascular disease. To understand this apparent paradox of increased inhibition of fibrinolysis (high PAI-1) combined with increased fibrinolytic activity (high D-dimer), we examined the relation between D-dimer, PAI-1 and the activator of fibrinolysis, tissue type plasminogen activator (t-PA) in subjects with varying severity of peripheral atherosclerosis. In 325 subjects selected from the Rotterdam Study, a cohort of 7983 men and women aged 55 years and over, the ankle to brachial systolic blood pressure ratio, t-PA antigen and activity, PAI-1 antigen and D-dimer were measured. T-PA antigen and t-PA activity were, independent from each other, increased with degree of atherosclerosis; t-PA antigen increased with 3.5 ng/ml (SE 1.7, p = 0.04) and t-PA activity with 0.46 IU/ml (0.20, p = 0.02) per unit decrease in ankle to brachial pressure ratio (i.e. increase in atherosclerosis). PAI-1 antigen was not related to atherosclerosis. More marked atherosclerosis was associated with increased D-dimer, mainly in subgroups with PAI-1 antigen below 50 ng/ml, t-PA antigen below 10 ng/ml, or t-PA activity above 1.5 IU/ml. In contrast to current beliefs, we found that only a fraction of the variation of t-PA antigen was due to the variation in circulating PAI-1 antigen. A slight positive association was observed between t-PA antigen and D-dimer. PAI-1 and t-PA activity were not associated with D-dimer concentration. In conclusion, in subjects with peripheral atherosclerosis PAI-1 antigen is not increased, but low PAI-1 levels (and possibly also low levels of t-PA antigen and high levels of t-PA activity) appear to be required to increase circulating D-dimer. This suggests that increased D-dimer levels in subjects with atherosclerosis do not reflect increased inhibition, but rather reflect increased fibrinolysis.

Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide.

Plasminogen activator inhibitor type 1 (PAI-1) is an important endogenous regulator of the fibrinolytic system. Reduction of PAI-1 activity has been shown to enhance dissolution of blood clots. Like other serpins, PAI-1 binds covalently to a target serine protease, thereby irreversibly inactivating the enzyme. During this process the exposed reactive-centre loop of PAI-1 is believed to undergo a conformational change becoming inserted into beta sheet A of the serpin. Incubation with peptides from the reactive-centre loop transform serpins into a substrate for their target protease. It has been hypothesised that these peptides bind to beta sheet A, thereby hindering the conformational rearrangement leading to loop insertion and formation of the stable serpin-protease complex. We report here the 1.95 A X-ray crystal structure of a complex of a glycosylated mutant of PAI-1, PAI-1-ala335Glu, with two molecules of the inhibitory reactive-centre loop peptide N-Ac-TVASS-NH2. Both bound peptide molecules are located between beta strands 3A and 5A of the serpin. The binding kinetics of the peptide inhibitor to immobilised PAI-1-Ala335Glu, as monitored by surface plasmon resonance, is consistent with there being two different binding sites. This is the first reported crystal structure of a complex formed between a serpin and a serpin inhibitor. The localisation of the inhibitory peptide in the complex strongly supports the theory that molecules binding in the space between beta strands 3A and 5A of a serpin are able to prevent insertion of the reactive-centre loop into beta sheet A, thereby abolishing the ability of the serpin to irreversibly inactivate its target enzyme. The characterisation of the two binding sites for the peptide inhibitor provides a solid foundation for computer-aided design of novel, low molecular weight PAI-1 inhibitors.

Clearance of tissue plasminogen activator (TPA) and TPA/plasminogen activator inhibitor type 1 (PAI-1) complex: relationship to elevated TPA antigen in patients with high PAI-1 activity levels.

To evaluate the effect of plasminogen activator inhibitor type 1 (PAI-1) levels on the clearance of total tissue plasminogen activator (TPA) antigen, we studied the clearance of active TPA and TPA/PAI-1 complex in subjects with low (181+/-109 pmol/L; n=7) and high (1166+/-322 pmol/L; n=4) baseline active PAI-1. A 5-microg/kg bolus of TPA was infused over a 15-second period followed by measurement of TPA activity, TPA antigen, TPA/PAI-1, TPA/C1 inhibitor, PAI-1 activity, and PAI-1 antigen over a 4-hour period. alpha-Phase clearance of total TPA antigen was faster in subjects with low PAI-1 (t(1/2) of 3.5+/-0.7 minutes) versus high PAI-1 (t(1/2) of 5.3+/-0.9 minutes) (P=.006). Clearance of all factors was best fit by a two-compartment pharmacokinetic model based on a computer-simulated human circulatory system. The average hepatic clearance fraction in the two-compartment model was greater for active TPA (89+/-10%, t(1/2) of 2.4+/-0.3 minutes) than for TPA/PAI-1 complex (48+/-17%, t(1/2) of 5.0+/-1.8 minutes) (P=.0006). Plasma clearance of active TPA was faster than clearance of TPA/PAI-1 complex. High levels of active PAI-1 converted more TPA into TPA/PAI-1 complex, effectively slowing the clearance of total TPA antigen and explaining in part why high levels of PAI-1 activity are associated with increases in total TPA antigen.

Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma

Plasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n = 11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in peripheral areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma.