PAI-1 Antigen Levels Research Articles

Fibrinogenolysis and fibrinolysis in vaccine-induced immune thrombocytopenia and thrombosis

BackgroundVaccine-induced immune thrombocytopenia and thrombosis (VITT) is a rare syndrome associated with adenoviral vector vaccines for COVID-19. The syndrome is characterized by thrombosis, anti-platelet factor 4 (PF4) antibodies, thrombocytopenia, high D-dimer, and hypofibrinogenemia. ObjectivesTo investigate abnormalities in fibrinolysis that contribute to the clinical features of VITT. MethodsPlasma samples from 18 suspected VITT cases were tested for anti-PF4 by ELISA and characterized as meeting criteria for VITT (11/18) or deemed unlikely (7/18; non-VITT). Antigen levels of PAI-1, factor XIII (FXIII), plasmin-α2antiplasmin (PAP), and inflammatory markers were quantified. Plasmin generation was quantified by chromogenic substrate. Western blotting was performed with antibodies to fibrinogen, FXIII-A, and plasminogen. ResultsVITT patients 10/11 had scores indicative of overt disseminated intravascular coagulation, while 0/7 non-VITT patients met the criteria. VITT patients had significantly higher levels of inflammatory markers, IL-1β, IL-6, IL-8, TNFα, and C-reactive protein. In VITT patients, both fibrinogen and FXIII levels were significantly lower, while PAP and tPA-mediated plasmin generation were higher compared to non-VITT patients. Evidence of fibrinogenolysis was observed in 9/11 VITT patients but not in non-VITT patients or healthy controls. Fibrinogen degradation products were apparent, with obvious cleavage of the fibrinogen α-chain. PAP complex was evident in those VITT patients with fibrinogenolysis, but not in non-VITT patients or healthy donors. ConclusionVITT patients show evidence of overt disseminated intravascular coagulation and fibrinogenolysis, mediated by dysregulated plasmin generation, as evidenced by increased PAP and plasmin generation. These observations are consistent with the clinical presentation of both thrombosis and bleeding in VITT.

Plasminogen activator inhibitor-2 and impaired fibrinolysis in pregnancy and sickle cell anemia

PurposeThis is the first study that aimed to determine antigen levels in plasma and genotypes of PAI-2 in pregnant and non-pregnant homozygous sickle cell anemia (SCA) patients.MethodsThe study subjects were all Bahraini females in the reproductive age group. The study population included 31 pregnant homozygous SS (SCA) patients. Three control groups were also studied to evaluate the effect of pregnancy and SCA on PAI-2 levels and fibrinolysis: (1) 31 healthy non-pregnant volunteers; (2) 31 cases of normal pregnancy; and (3) 20 non-pregnant SCA patients. Pregnancies were screened in the second (TM2) and third (TM3) trimesters. Global coagulation, fibrinolysis rate (euglobulin clot lysis time, ECLT), PAI-2 antigen (ELISA), and PAI-2 Ser(413)/Cys polymorphism (restriction fragment length polymorphism analysis) were determined.ResultsFeto-maternal complications were documented in both pregnancy groups. PAI-2 antigen levels were undetectable in the non-pregnant groups, but was quantifiable in both pregnant groups. Impaired fibrinolysis rate and rising PAI-2 levels with progression of pregnancy were observed in both healthy and SCA subjects. These changes were more prominent in SCA, although the rise in ECLT was less steep and PAI-2 antigen levels were not significantly different compared to normal pregnancy in the third trimester. No correlation was observed between PAI-2 genotypes and plasma antigen levels. Also, no significant difference in feto-maternal complications was found in normal (n = 25) versus SCA pregnant patients (n = 30).ConclusionsThese observations suggest that with progression of pregnancy, increasing PAI-2 levels contribute to the hypercoagulable state, particularly in SCA patients.

Clinical impact of the PAI-1 4G/5G polymorphism in Chinese patients with venous thromboembolism

BackgroundVenous thromboembolism (VTE) is a life-threatening cardiovascular syndrome that characterized by the imbalance of hemostasis and thrombosis and the formation of thrombi in the blood vessels. The aim of this study was to elucidate the clinical impact of the PAI-1 4G/5G polymorphism in Chinese patients with VTE.MethodsA total of 169 subjects (89 VTE, 10 hyperbilirubinemia, 10 hyperlipidemia and 60 healthy controls) were recruited at Peking Union Medical College Hospital. The accuracy of the TaqMan-MGB RT-PCR method for detecting F5 G1691A (FVL) and PAI-1 4G/5G polymorphisms was evaluated by using sequencing method as the gold standard. Besides, the association of the PAI-1 4G/5G polymorphism with susceptibility, treatment efficacy and recurrence status of VTE in Chinese population were explored. Eventually, the plasma PAI-1 antigen levels and PAI-1 4G/5G polymorphisms were determined on additional 64 subjects (32 VTE and 32 healthy controls) simultaneously.ResultsThe TaqMan-MGB RT-PCR method was proven to be highly accurate in determining the FVL and PAI-1 4G/5G polymorphisms without interference from bilirubin and lipids in the samples. No obvious correlation of the PAI-1 4G/5G polymorphism with VTE was observed in our study by using five genetic models (allele, genotype, dominant, recessive and additive). Additionally, we also observed that individuals with the 4G/5G genotype had lower neutrophil counts and neutrophil-to-lymphocyte ratio (NLR) than the 5G/5G genotype. Furthermore, we found that the patients with the 5G/5G genotype were more likely to achieve complete recanalization compared to the 4G/4G genotype. In addition, individuals carrying the 5G/5G genotype were more likely to develop a recurrence-free status as compared to individuals with the 4G/4G or 4G/5G genotypes. PAI-1 antigen levels in the VTE group were significantly higher than those in the HC group. However, there was no significant difference in the antigen levels of PAI-1 among subjects carrying various genotypes in the VTE group or HC group.ConclusionThe PAI-1 4G/5G polymorphism has potential value in assessing the prognosis of Chinese patients with VTE. Our study has laid the foundation for the application of PAI-1 4G/5G polymorphism in the personalized management and monitoring of patients with VTE.

Fibrinolytic Dysregulation Contributes to the Hypercoagulable State in Pulmonary Embolism Patients

Introduction: The multifactorial and complex pathophysiology of pulmonary embolism (PE) involves dysregulation of hemostatic system including fibrinolytic imbalance, endothelial compromise and generation of thrombotic mediators, along with cellular defects and hemodynamic derangement. It is estimated that 6000 cases of pulmonary embolism and DVT are reported yearly of which 1000 result in death. PE evolves from a pre-existing venous thrombus, obstructing pulmonary artery leading to pulmonary failure. The progression of clot formation results in increase in right ventricular pressure, systemic hypoperfusion, hypoxemia ischemia and eventually multi organ failure. Fibrinolytic dysregulation and generation of procoagulant mediators result in increased clot burden and lead to adverse outcomes. Profiling of the component of the fibrinolytic system for such mediators as the activators and inhibitors of fibrinolysis, vWF and cellular microparticles may provide additional insights into the pathogenesis of PE. The purpose of this study is to profile plasma samples from PE patients for various mediators of hemostatic dysregulation which contribute to the fibrinolytic dysregulation in PE patients.Material & Method: Seventy-eight patients of 18 years or older age, were included in this study through enrolment conducted in conjunction with an ongoing IRB approved project by the pulmonary embolism response team (PERT) registry. Diagnosis of PE was confirmed by computed tomography (CT), angiography or ventilation perfusion imaging. Blood samples were drawn with in 24 hours of confirmed diagnosis of acute PE in 3.2% sodium citrate tubes at the time of diagnosis, processed for platelet poor plasma and frozen. Control plasma samples were obtained from 50 healthy individuals. Both the patient and control plasma samples were analyzed for D-Dimer, PAI-1 antigen, tPA, TAFI-a, protein C, alpha-2 antiplasmin, microparticles and vWF using ELISA methods (Hypen Biomedical, Franklin, Ohio). A functional PAI-1 chromogenic substrate method and an ELISA based uPA antigen method was used (Biomedica, Halifax, Nova Scotia, Canada). Circulating levels of each of the individual biomarkers were compared to normal plasma, descriptive statistics including non-parametric Mann-Whitney t-test and spearman correlation coefficient analysis was carried out by using GraphPad Prism software. Percent Increase from the normal mean was calculated for each individual parameter. p value <0.05 was considered statistically significant.Result: In comparison to normal group, the PE patients exhibited varying levels of increase in the D-Dimer, tPA, uPA, PAI-1 antigen, PAI-1 functional, TAFI antigen, microparticles and vWF, whereas decreased levels were noted for protein C and alpha-2 antiplasmin as shown on Figure 1. D-Dimer showed the most pronounced increase (368.2-fold) followed by tPA (165.9-fold), PAI-1 antigen (3.91-fold), microparticles (2.14-fold), vWF (1.83-fold), uPA (0.5-fold), TAFI antigen (0.40-fold) and PAI-1 functional (0.34-fold). In correlation analysis, D-Dimer negatively correlated with TAFI antigen (r= -0.3106) and alpha-2 antiplasmin (r= -0.3056), functional PAI-1 levels positively correlated with tPA (r= 0.3117) and protein C (r= 0.4059), tPA negatively correlated with TAFI antigen (r= -0.4601), alpha-2 antiplasmin (r= -0.2460) and positively correlated with microparticles (r= 0.2456) , TAFI antigen positively correlated with protein C (r= 0.3612), alpha-2 antiplasmin (r= 0.3899) and negatively correlated with microparticles (r= -0.2470) whereas protein C showed a negative correlation with vWF (r= -0.2838).Conclusion: The markedly increased D-Dimer levels suggest increase clot burden in PE patients. tPA antigen is also markedly increased, whereas the uPA showed modest increase which may suggest compensatory upregulation, PAI-1 antigen levels were much higher in comparison to PAI-1 functional activity, suggesting consumption of this inhibitor. Decreased alpha-2 antiplasmin and protein C levels also suggest consumption. Increased TAFI antigen contribute to the clot resistant to fibrinolysis. Cellular consumption and endothelial dysfunction results in increased in vWF and microparticles promoting thrombogenesis. Measurement of these biomarkers may be useful in the understanding of the pathogenesis, risk stratification and clinical management of PE patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hemostatic state of children with type 1 diabetes.

PurposeHyperglycemia is one of the factors responsible for the molecular alterations that modify hemostasis. The aim of this study was to determine the levels of circulating molecules that have a prothrombotic impact on the child and adolescent population with type 1 diabetes mellitus.MethodsThere were 35 patients with type 1 diabetes mellitus (11.0±2.5 years of age and a median 3.7±2.0 years of the disease) with no vascular complications and 20 healthy controls with similar age, sex, and body mass index included in the study. The evaluated parameters were fibrinogen, plasminogen activator inhibitor-1 (PAI-1), von Willebrand factor antigen, and standard coagulation tests (platelet count, prothrombin time, and activated partial thromboplastin time). Glycemic control was evaluated by hemoglobin A1c and fasting blood glucose tests, and the presence of retinopathy and nephropathy was ruled out. The data obtained were analyzed by IBM SPSS Statistics ver. 20.0 and expressed as mean±standard deviation. The Pearson correlation coefficient was applied to investigate correlations between variables.ResultsDiabetic patients showed significantly higher levels of fibrinogen (308±66 mg/dL vs. 246±18 mg/dL, P=0.0001), PAI-1 (41.6±12 ng/mL vs. 11.7±1.0 ng/mL, P=0.0001), and von Willebrand factor antigen (284%±55% vs. 121%±19%, P=0.0001). However, standard coagulation tests did not show differences between the 2 groups. PAI-1 was correlated with glycemia, hemoglobin A1c, fibrinogen, and von Willebrand factor antigen.ConclusionsElevated levels of fibrinogen, PAI-1, and von Willebrand factor antigen were found in the pediatric and adolescent population with type 1 diabetes mellitus, which suggests a prothrombotic state.

Epigenetic modulators combination with chemotherapy in breast cancer cells.

Despite the concerning adverse effects on tumour development, epigenetic drugs are very promising in cancer treatment. The aim of this study was to compare the differential effects of standard chemotherapy regimens (FEC: 5-fluorouracil plus epirubicine plus cyclophosphamide) in combination with epigenetic modulators (decitabine, valproic acid): (a) on gene methylation levels of selected tumour biomarkers (LINE-1, uPA, PAI-1, DAPK); (b) their expression status (uPA and PAI-1); (c) differentiation status (5meC and H3K27me3). Furthermore, cell survival as well as changes concerning the invasion capacity were monitored in cell culture models of breast cancer (MCF-7, MDA-MB-231). A significant overall decrease of cell survival was observed in the FEC-containing combination therapies for both cell lines. Methylation results showed a general tendency towards increased demethylation of the uPA and PAI-1 gene promoters for the MCF-7 cells, as well as the proapoptotic DAPK gene in the treatment regimens for both cell lines. The uPA and PAI-1 antigen levels were mainly increased in the supernatant of FEC-only treated MDA-MB-231 cells. DAC-only treatment induced an increase of secreted uPA protein in MCF-7 cell culture, while most of the VPA-containing regimens also induced uPA and PAI-1 expression in MCF-7 cell fractions. Epigenetically active substances can also induce a re-differentiation in tumour cells, as shown by 5meC, H3K27me3 applying ICC. SIGNIFICANCE OF THE STUDY: Epigenetic modulators especially in the highly undifferentiated and highly malignant MDA-MB-231 tumour cells significantly reduced tumour malignancy thus; further clinical studies applying specific combination therapies with epigenetic modulators may be warranted.

Prognostic significance of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) in patients with primary invasive ductal breast carcinoma - a 7.5-year follow-up study.

Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) are key molecules in pericellular proteolysis, a process that plays an important role in tumor invasion and metastasis. In the current study we investigated the prognostic significance of uPA and PAI-1 in primary invasive breast cancer. uPA and PAI-1 antigen levels were determined by enzyme-linked immunosorbent assay in cytosols of 177 invasive ductal carcinoma specimens. The prognostic significance of uPA and PAI-1 was assessed for overall survival. The median follow-up time was 90 months. In univariate analysis, both uPA (third versus first tertile range of values; P = 0.02; HR = 2.08) and PAI-1 (third versus first tertile; P = 0.0007; HR = 3.1) were significant prognostic markers for overall survival. In multivariate analysis only nodal status (N2 vs N0; P = 0.0001; HR = 3.94) and PAI-1 (third versus first tertile; P = 0.004; HR = 3.05) remained significant independent prognostic factors. Both uPA and PAI-1 were correlated with established prognostic markers including histological grade, tumor size and Nottingham index. Our study with a 7.5-year follow-up confirmed the relation between elevated uPA and PAI-1 values and an aggressive course of invasive breast cancer. The prognostic significance of PAI-1 as an independent marker was proved for the overall group of breast cancer patients and the subgroup of node-positive patients.

Plasminogen activator inhibitor and the risk of cardiovascular disease: The Framingham Heart Study

IntroductionAlthough plasminogen activator inhibitor (PAI-1) plays a key regulatory role in fibrinolysis, it has not been clearly shown to independently predict cardiovascular disease (CVD) among individuals without prior CVD. We investigated, in the Framingham Heart Study offspring cohort, whether PAI-1 predicted CVD risk among individuals without prior CVD. MethodsPlasma PAI-1 antigen and tissue plasminogen activator (TPA) antigen were measured in 3203 subjects without prior CVD between 1991 and 1995; average follow-up of 10years. PAI-1 was remeasured 4years after baseline, to determine the effect of serial change on risk. ResultsPAI-1 levels (mean±SD) were 29.1ng/ml (19.2) versus 22.1 (16.5) for those and without incident CVD; p<0.001, and TPA levels were 12.0ng/ml (5.7) versus 9.0 (4.7); p<0.001. PAI-1 and TPA antigen levels had a strong unadjusted linear relation with incident CVD (p<0.001). After adjustment for conventional risk factors, the hazard ratios (HRs) for higher quartiles of PAI-1, compared with the lowest, were 1.9, 1.9, 2.6 (linear trend p=0.006), and 1.6, 1.6, 2.9 (p<0.001) for TPA antigen. The adjusted HRs for increasing quartiles of serial change in PAI-1 at 4years, compared with the lowest, were 0.9, 0.8, 1.3 (p=0.050). C statistic assessment showed that adding PAI-1 or TPA to conventional risk factors resulted in small increases in discrimination and modest reclassification of risk, which was statistically significant for TPA (net reclassification 6.8%, p=0.037) but not PAI-1 (4.8%, p=0.113). ConclusionPAI-1 and TPA antigen levels are predictive of CVD events after accounting for established risk factors. A serial increase in PAI-1 is associated with a further increase in risk. These findings support the importance of fibrinolytic potential in CVD.

Profibrinolytic effect of the epigenetic modifier valproic acid in man.

AimsThe aim of the study was to test if pharmacological intervention by valproic acid (VPA) treatment can modulate the fibrinolytic system in man, by means of increased acute release capacity of tissue plasminogen activator (t-PA) as well as an altered t-PA/Plasminogen activator inhibitor -1 (PAI-1) balance. Recent data from in vitro research demonstrate that the fibrinolytic system is epigenetically regulated mainly by histone deacetylase (HDAC) inhibitors. HDAC inhibitors, including VPA markedly upregulate t-PA gene expression in vitro.Methods and ResultsThe trial had a cross-over design where healthy men (n = 10), were treated with VPA (Ergenyl Retard) 500 mg depot tablets twice daily for 2 weeks. Capacity for stimulated t-PA release was assessed in the perfused-forearm model using intra-brachial Substance P infusion and venous occlusion plethysmography. Each subject was investigated twice, untreated and after VPA treatment, with 5 weeks wash-out in-between. VPA treatment resulted in considerably decreased levels of circulating PAI-1 antigen from 22.2 (4.6) to 10.8 (2.1) ng/ml (p<0.05). It slightly decreased the levels of circulating venous t-PA antigen (p<0.05), and the t-PA:PAI-1 antigen ratio increased (p<0.01). Substance P infusion resulted in an increase in forearm blood flow (FBF) on both occasions (p<0.0001 for both). The acute t-PA release in response to Substance P was not affected by VPA (p = ns).ConclusionValproic acid treatment lowers plasma PAI-1 antigen levels and changes the fibrinolytic balance measured as t-PA/PAI-1 ratio in a profibrinolytic direction. This may in part explain the reduction in incidence of myocardial infarctions by VPA treatment observed in recent pharmacoepidemiological studies.Trial RegistrationThe EU Clinical Trials Register 2009-011723-31

Ki-67 as an independent prognostic factor in an unselected cohort of patients with ovarian cancer: Results of an explorative, retrospective study

The identification of prognostic markers has clinical implications in epithelial ovarian carcinoma (EOC). Here, we studied markers for proliferation (Ki-67), endocrine regulation [progesterone receptor (PR), estrogen receptor (ER)], and invasion [urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1)]. All patients with available follow-up information and EOC tissue, who were treated at our institution between 1997 and 2004, were enrolled in the present study. Expression of Ki-67, PR and ER was determined by immunohistochemical analyses. uPA and PAI-1 antigen levels were determined using enzyme‑linked immunosorbent assays. One hundred and eight patients entered the present study. The median follow-up time was 43.3 (range 11.4-68.0) months. In multivariable Cox regression analyses, Ki-67 expression showed an independent negative impact on disease-free survival (DFS) and overall survival (OS) [hazard ratio (HR) for DFS, 11.5; 95% confidence interval (CI), 2.64-49.7; p=0.001 and HR for OS, 21.2; 95% CI, 9.9-113.1; p<0.001]. After cut-off optimization, PR expression showed an independent positive impact on prognosis (HR for DFS, 0.15; 95% CI, 0.03-0.68; p=0.014 and HR for OS, 0.13; 95% CI, 0.03‑0.68; p=0.016). Furthermore, postoperative residual tumor burden and completeness of chemotherapy determined the prognosis. ER, uPA and PAI-1 were not associated with survival. PR and ER, and postoperative residual tumor burden and tumor stage showed a strong correlation in an explorative Spearman's rank correlation coefficient (rho=0.759 and rho=0.426, respectively). Ki-67 and cut-off optimized PR are independently associated with the prognosis of EOC. Further prospective studies are warranted to confirm these associations and to elucidate the underlying mechanisms.

Functionally stable plasminogen activator inhibitor-1 in a family with cardiovascular disease and vitiligo

Vitiligo is a common skin condition with a complex pathophysiology characterized by the lack of pigmentation due to melanocyte degeneration. In this study, we investigated PAI-1 antigen (Ag) and activity levels in a 34 year old male with extensive vascular disease, alopecia areata and vitiligo. Fasting PAI-1 Ag and activity levels were measured at 9 a.m. in the subject and family members. Both PAI-1 Ag (67 ± 38 vs. 18.6 ± 6.5 ng/ml, P < 0.001) and specific activity (15.8 ± 10.0 vs. 7.6 ± 6.0 IU/pmol, P < 0.04) levels of PAI-1 were moderately elevated in subjects compared to the controls. PAI-1 kinetic studies demonstrated a markedly enhanced stability of plasma PAI-1 activity in the family members. Specific activity at 16 h was significantly higher than expected activity levels (0.078 ± 0.072 vs. 0.001 ± 0.001 IU/ng/ml, P < 0.001). While the exact mechanism of increased stability of PAI-1 activity in vitiligo is not known, it is likely due to post-translational modifications or increased binding affinity for a stabilizing cofactor. In conclusion, enhanced stability of PAI-1 may contribute to the pathophysiology of vascular disease and associated melanocyte degeneration. Systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative to the near orphan status for vitiligo drug development.

GW24-e2512 A study on plasma PAI-1 and promoter-844 G- A,-675 4G/SG polymorphism of CHD

ObjectivesTo investigate the concentration of plasma plasminogen activator inhibitor-1 and the promoter-844G-A substitution,-675 4G/5G polymorphism in patients with CHD and control.Methods293 patients with CHD (UAP group andAMIgroup)and 173 human in...

4G/5G Polymorphism of the Plasminogen Activator Inhibitor-1 Gene Is Associated with Multiple Organ Dysfunction in Critically Ill Patients

Objective: Impaired fibrinolysis is associated with a higher incidence of both multiple organ dysfunction and mortality in the intensive care unit (ICU). Plasminogen activator inhibitor (PAI)-1 is the chief inhibitor of fibrinolysis. We investigated the influence of the 4G/5G polymorphism (rs1799768) of the PAI-1 gene on the plasma PAI-1 level and the outcome of critically ill patients. Methods: In 41 consecutive patients admitted to the ICU, PAI-1 gene polymorphism was assessed, plasma PAI-1 and arterial lactate concentrations were measured and clinical severity scores were recorded. Results: Homozygotes for the 4G allele had higher plasma levels of PAI-1 antigen. The mean ± SD PAI-1 antigen level was 193.31 ± 167.93 ng/ml for the 4G/4G genotype, 100.67 ± 114.16 ng/ml for the 4G/5G genotype and 0.43 ± 0.53 ng/ml for the 5G/5G genotype. There was a significant correlation between plasma PAI-1 and arterial lactate concentrations, as well as between PAI-1 and severity scores. The mortality rate was 63, 33 and 0% for patients with the 4G/4G, 4G/5G and 5G/5G genotypes, respectively. Conclusions: These results demonstrate that the 4G/5G polymorphism of the PAI-1 gene affects the plasma PAI-1 concentration, which could impair fibrinolysis and cause organ failure, and thus the presence of the 4G allele increases the risk of death.

Prognostic significance of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) in patients with primary invasive ductal breast carcinoma – a 7.5-year follow-up study

Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) are key molecules in pericellular proteolysis, a process that plays an important role in tumor invasion and metastasis. In the current study we investigated the prognostic significance of uPA and PAI-1 in primary invasive breast cancer. uPA and PAI-1 antigen levels were determined by enzyme-linked immunosorbent assay in cytosols of 177 invasive ductal carcinoma specimens. The prognostic significance of uPA and PAI-1 was assessed for overall survival. The median follow-up time was 90 months. In univariate analysis, both uPA (third versus first tertile range of values; P = 0.02; HR = 2.08) and PAI-1 (third versus first tertile; P = 0.0007; HR = 3.1) were significant prognostic markers for overall survival. In multivariate analysis only nodal status (N2 vs N0; P = 0.0001; HR = 3.94) and PAI-1 (third versus first tertile; P = 0.004; HR = 3.05) remained significant independent prognostic factors. Both uPA and PAI-1 were correlated with established prognostic markers including histological grade, tumor size and Nottingham index. Our study with a 7.5-year follow-up confirmed the relation between elevated uPA and PAI-1 values and an aggressive course of invasive breast cancer. The prognostic significance of PAI-1 as an independent marker was proved for the overall group of breast cancer patients and the subgroup of node-positive patients.

Role of fibrinolytic parameters and plasminogen activator inhibitor 1 (PAI-1) promoter polymorphism on premature atherosclerosis in SLE patients

Premature atherosclerosis has been recognized as a major co-morbid condition in systemic lupus erythematosus (SLE). In this study, we aimed to investigate the effect of tPA (tissue plasminogen activator) and PAI-1 (plasminogen activator inhibitor) antigen concentrations and 4G/5G polymorphism of the PAI-1 gene on the development of atherosclerosis in SLE patients. One hundred and six SLE patients, 28 Takayasu arteritis (TA) patients and 98 healthy control subjects (HCs) were studied. PAI-1 and tPA antigen levels were measured by ELISA method. PAI-1 gene polymorphism was determined by using allele-specific PCR method. SLE patients had a significantly higher frequency (22.6%) of plaque (p = 0.01) and higher IMT (p=0.04) compared with HCs respectively. Only age at disease onset was associated with plaque formation in multivariate regression analysis (p = 0.001). Plasma tPA ag levels in SLE patients were significantly higher compared with HCs (p = 0.005) and PAI-1 ag levels were significantly higher compared with TA patients (p = 0.03). There were no significant differences between study groups in both genotype distribution and allele frequencies of PAI-1 gene, but SLE patients with 4G/4G genotype had higher IMT (p = 0.02) calcium scoring (p = 0.006) compared with 4G5G/5G5G genotypes. The present study suggests that measuring fibrinolytic parameters would have little additional benefit beyond traditional and novel risk factors in predicting coronary artery disease (CAD).

Co-detection of members of the urokinase plasminogen activator system in tumour tissue and serum correlates with a poor prognosis for soft-tissue sarcoma patients

Background:The urokinase plasminogen activator (uPA) system is one of the best-investigated protease systems, both under physiological and pathological conditions, including various types of cancer. However, effects of co-expression of members of the uPA system in soft-tissue sarcoma (STS) patients at the protein level in both tumour tissue and serum have not been investigated yet.Methods:We examined 82 STS patients for protein levels of uPA, PAI-1and uPAR in tumour tissue and serum by ELISA.Results:A significant correlation between high antigen levels of uPA, PAI-1 or uPAR in tumour tissue, and of uPAR in serum, with poor outcome of STS patients was found for the first time. Most strikingly, we observed an additive effect of combined uPA, PAI-1 or uPAR levels in tumour tissue extracts with uPAR levels in serum on patients’ prognosis. High uPA/uPAR, PAI-1/uPAR and uPAR/uPAR antigen levels in tumour tissue/serum were associated with a 5.9-fold, 5.8-fold and 6.2-fold increased risk of tumour-related death (P=0.003, 0.001 and 0.002, respectively) compared with those patients who displayed low levels of the respective marker combination.Conclusion:As expression of members of the uPA system in tumour tissue and serum is additively correlated with prognosis of STS patients, our results suggest that combinations of these biomarkers can identify STS patients with a higher risk of tumour-related death.

PAI-1 donor polymorphism influences long-term kidney graft survival

The type 1 plasminogen activator inhibitor (PAI-1) is involved in the development of fibrosis, and its intrarenal expression is increased in interstitial fibrosis and tubular atrophy (IFTA). Moreover, a 4G/5G polymorphism of the PAI-1 gene has been described associating 4G haplotype with higher PAI-1 plasma activity. We investigated the relationship between the donor and recipient PAI-1 polymorphism and kidney graft survival. The PAI-1 genotype was determined for both the 304 donors and the 337 corresponding recipients. In recipients, PAI-1 antigen levels were also determined. We compared 4G/4G donors versus donors with other genotypes. Donor or recipient genotype did not influence the PAI-1 plasma level in recipients. Actuarial kidney graft survival was significantly reduced in the 4G/4G donor group (107 months versus 147.5 months, P = 0.013), while recipient PAI-1 genotype did not show any influence on graft survival. Moreover, graft loss due to IFTA proved significantly higher in the 4G/4G donor group (13% versus 6%, P = 0.03). Multivariate analysis showed that the significant independent variables associated with graft loss were the donor 4G/4G genotype, acute clinical rejection and donor age. Our study suggests that donor PAI-1 polymorphism influences kidney graft survival and that the donor 4G/4G genotype is an independent risk factor for graft loss. Prospective studies are needed to confirm these results.

PAI-1 levels predict response to fractionated irradiation in 10 human squamous cell carcinoma lines of the head and neck

Background and purpose To investigate the relationships between hypoxia, VEGF and components of the plasminogen activation system (PAS) and to determine their influence on local tumour control after fractionated radiotherapy. Material and methods Ten cell lines derived from human squamous cell carcinomas of the head and neck (SCCHN) were investigated in vitro and used to generate xenograft tumours. The pimonidazole hypoxic fraction in the total tumour area (pHF tot) was used to measure hypoxia in pre-treatment tumours and the local tumour control (TCD 50) was used as the functional endpoint in vivo. For in vitro experiments, cells were cultured for 24 h under either normoxic or mild hypoxic (∼0.66% O 2) conditions. VEGF, PAI-1 and uPA antigen levels were determined by ELISA and uPA activity by an activity assay kit. Results Of all the factors investigated, only PAI-1 expression correlated with TCD 50 ( r = 0.80, p = 0.010) and was significantly higher ( p = 0.001) in more hypoxic than in less hypoxic tumours. Accordingly, PAI-1 secretion was significantly induced (2.4×) by in vitro hypoxia. Conclusions These results suggest that pre-treatment PAI-1 levels are higher in more hypoxic tumours and can predict the response to fractionated irradiation in SCCHN.

Heating of carbon dioxide during insufflation alters the peritoneal fibrinolytic response to laparoscopic surgery

Laparoscopic surgery is evolving rapidly. It involves the creation of a pneumoperitoneum, mostly using carbon dioxide. Cooling of the peritoneum, due to insufflation, might traumatize the peritoneum and disturb peritoneal fibrinolysis, important in peritoneal healing processes. The current study was performed to elucidate the effects of the temperature of insufflation gas on the peritoneal fibrinolytic response to laparoscopic surgery. Thirty patients scheduled for laparoscopic cholecystectomy were randomized in two groups: one group in which the pneumoperitoneum was created with carbon dioxide at room temperature, and one wherein carbon dioxide at body temperature was used. Peritoneal biopsies were taken at the start and at the end of surgery. Tissue concentrations of tPA antigen, tPA activity, uPA antigen, and PAI-1 antigen were measured using ELISA techniques. Peritoneal PAI-1 antigen levels were significantly higher at the end of the procedure in patients operated with carbon dioxide at room temperature (p < .05). A slight, but not significant, decrease in tPA antigen and activity was observed in both groups during the procedure. Peritoneal concentrations of uPa antigen did not change during the procedure. The temperature of carbon dioxide used for insufflation of the abdominal cavity affects peritoneal biology. Cooling of the peritoneum by unheated carbon dioxide causes increased peritoneal PAI-1 levels, important in peritoneal healing processes.

Plasminogen activator inhibitor 1 expression in platelets is not influenced by the 4G/5G promoter polymorphism

In the present study we investigated the influence of the 4G/5G promoter polymorphism of the PAI-1 gene on the levels of PAI-1 mRNA and protein in platelets. After a screening of healthy male subjects, thirty-eight subjects homozygote for either the 4G or 5G allele were investigated. mRNA levels were quantified by real-time PCR and PAI-1 antigen in platelets and plasma was analysed by ELISA. The platelet PAI-1 mRNA levels correlated significantly with the PAI-1 antigen content, but there was no association between the polymorphism and mRNA levels, or protein levels in platelets. Also, plasma levels of PAI-1 antigen were not associated with homozygosity of the 4G/5G polymorphism, but as expected BMI and triglycerides emerged as significant predictors of plasma PAI-1 levels. The importance of the 4G/5G polymorphism on PAI-1 levels is controversial and the present study shows that although levels of platelet mRNA are related to its content of PAI-1 protein, there is no association between the 4G/5G promoter polymorphism and platelet PAI-1 mRNA or protein expression.