Higher Plasminogen Activator Inhibitor-1 Research Articles

Coronary thrombus in 34-year-old female patient with 4G/4G polymorphism in the PAI-1 gene

Genetic factors and hypofibrinolytic state may contribute to the likelihood of developing in myocardial infarction (MI) in young women rather than traditional risk factors. High plasminogen-activator inhibitor-1 (PAI-1) level and PAI-1 gene polymorphism have been shown to be associated with thrombotic events such as myocardial infarction, deep venous thrombosis, and stroke. We determined 4G/4G polymorphism in a 34-year-old female patient with subacute anterior myocardial infarction and coronary thrombus in left anterior descending artery on coronary angiogram.

Associations of Serum 25-Hydroxyvitamin D With Hemostatic and Inflammatory Biomarkers in the Multi-Ethnic Study of Atherosclerosis.

Mechanisms explaining documented associations of 25-hydroxyvitamin D [25(OH)D] deficiency with increased risks of cardiovascular disease (CVD) and venous thromboembolism may relate to adverse hemostatic and inflammatory responses. To evaluate whether 25(OH)D deficiency is associated with a prothrombotic and proinflammatory biological profile. Cross-sectional analyses. The Multi-Ethnic Study of Atherosclerosis, a multicenter prospective cohort of American adults. Up to 6554 adults free of CVD. Ten hemostatic biomarkers (D-dimer, fibrinogen, factor VIII, plasmin-antiplasmin, and homocysteine [n = 6443]; von Willebrand factor, soluble tissue factor, plasminogen activator inhibitor-1 (PAI-1), total tissue factor pathway inhibitor (TFPI), and soluble thrombomodulin [n = 814]), and three inflammatory biomarkers (IL-6, C-reactive protein [n = 6443], and TNF-α soluble receptor [n = 3802]). Among 6443 subjects (46.6% men; mean age, 62.1 years; mean body mass index, 28.3 kg/m(2)) of White (37.8%), Black (27.2%), Chinese (12.2%), and Hispanic (21.8%) race/ethnicity, mean 25(OH)D was 25.3 ng/mL. After multiple adjustment, 25(OH)D concentrations were associated with concentrations of IL-6 and homocysteine and also with concentrations of PAI-1 and TFPI: per 10 ng/mL decrement in 25(OH)D, 5.1% higher IL-6 (95% confidence interval [CI], 3.4-6.9; P < .001); 3.7% higher homocysteine (95% CI, 3.0-4.3; P < .001); 7.0% higher PAI-1 (95% CI, 0.9-13.6; P = .025); and 2.1% higher TFPI (95% CI, 0.0-4.2; P = .047), without racial/ethnic heterogeneity. No significant associations were observed for other hemostatic and inflammatory biomarkers. Increased inflammation as reflected by higher circulating IL-6 and increased homocysteine concentrations may represent mechanisms linking 25(OH)D deficiency to greater risks of CVD and perhaps venous thromboembolism. Low concentrations of 25(OH)D were also associated with PAI-1 and TFPI concentrations, but not with other hemostatic biomarkers.

Prognostic significance of uPA/PAI-1 level, HER2 status, and traditional histologic factors for survival in node-negative breast cancer patients.

BackgroundThe association of HER2 status with urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) levels raises the question whether uPA/PAI-1 level carries additional clinically relevant prognostic information independently from HER2 status. The aim of our study was to compare the prognostic value of uPA/PAI-1 level, HER2 status, and traditional prognostic factors for survival in node-negative breast cancer patients.Patients and methodsA retrospective analysis of 858 node-negative breast cancer patients treated in Maribor University Clinical Center, Slovenia, in the years 2000–2009 was performed. Data were obtained from patient medical records. The median follow-up time was 100 months. Univariate and multivariate analyses of disease-free (DFS) and overall survival (OS) were performed using the Cox regression and the Cox proportional hazards model.ResultsIn univariate analysis, age, tumor size, grade, lymphovascular invasion, HER2 status and UPA/PAI-1 level were associated with DFS, and age, tumor size, grade, and uPA/PAI-1 level were associated with OS. In the multivariate model, the most important determinants of DFS were age, estrogen receptor status and uPA/PAI-1 level, and the most important factors for OS were patient age and tumor grade. The HR for death from any cause in the multivariate model was 1.98 (95% CI 0.83–4.76) for patients with high uPA and/or PAI-1 compared to patients with both values low.ConclusionsuPA/PAI-1 level clearly carries an independent prognostic value regardless of HER2 status in node-negative breast cancer and could be used in addition to HER2 and other markers to guide clinical decisions in this setting.

The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues

Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.

Clinical and Laboratory Predictors of Major Adverse Cardiac Events in Patients with Ischemic Heart Disease Following Elective Percutaneous Coronary Intervention

Background. Despite recent advances in stent design and constantly improving protective pharmacological strategies, complications and adverse events following percutaneous coronary interventions (PCI) are still major factors influencing morbidity and mortality. Therefore, predicting secondary vascular occlusions represents an unmet medical need. Aim. To triage clinical and laboratory predictors of major adverse clinical events (MACE) following coronary stenting. Material and methods. This was a prospective, case-controlled, single-center study, which included 94 consecutive patients with documented ischemic heart disease (IHD) who underwent PCI with drug-eluting stent implantation. All patients received dual antiplatelet therapy with acetyl salicylic acid and clopidogrel. Numerous clinical characteristics and laboratory biomarkers were assessed before stenting, as well as CYP2C19 genotyping after patient’s discharge and were correlated with poststenting MACE over the mean follow-up of 28 months. MACE included death, nonfatal myocardial infarction, target vessel revascularisation, stroke, stent thrombosis, angina recurrence and in-stent restenosis. Results. Twenty-three patients experienced MACE. According to univariate regression analysis we found following MACE predictors after PCI: diabetes mellitus (p=0.049), P2Y12 Reaction Units (PRU) according to VerifyNow® (p=0.01), number of stented arteries more than 2 (p=0.01), number of implanted stents more than 2 (p=0.01), baseline levels of plasminogen activator inhibitor-1 (PAI-1) (p=0.03) and von Willebrand activity (vWF) (p=0.01). Using multivariate analysis we demonstrated that concomitant diabetes mellitus, PRU ≥202, PAI-1 level ≥75.95 ng/ml, von Willebrand factor activity ≥155.15% are independent predictors of adverse cardiac events after PCI in stable IHD patients. Other clinical characteristics and laboratory indices, including CYP2C19*2 carriage, showed no significant impact on outcomes after elective PCI. Conclusions. Background diabetes mellitus, high on-treatment platelet reactivity (according to VerifyNow®), PAI-1 and vWF presenting activity may be useful for MACE prediction over 28 months of follow-up after PCI with drug-eluting stent implantation.

Association of plasminogen activator inhibitor-1 gene polymorphism and type 2 diabetic nephropathy

Background: We investigated the relationship between plasminogen activator inhibitor-1 (PAI-1) 4G/5G insertion/deletion polymorphism and prevalence of diabetic nephropathy (DN) in Chinese patients. Methods: A total of 107 patients with type 2 diabetes were randomly recruited in the study, and 102 healthy subjects were selected as Control. Patients were divided into three groups according to their urinary albumin–creatinine ratio (UACR). Group A (n = 44), had patients without DN (serum creatinine <106 µmol/L and UACR <30 µg/mg); Group B (n = 30), had patients with micro-albuminuria (UACR 30–299 µg/mg), and Group C (n = 33), had patients with macro-albuminuria (UACR ≥300 µg/mg and creatinine <200 µmol/L). Plasma level of PAI-1 was measured by ELISA. PAI-1 polymorphism was determined by a polymerase chain reaction (PCR) method and DNA sequencing. Results: (1) The plasma PAI-1 levels of group A (60.39 ± 17.01 ng/L), group B (68.76 ± 17.81 ng/L) and group C and (68.63 ± 18.30 ng/L) are higher than that of controls (46.26 ± 26.04 ng/L); (2) Patients with genotype 4G/4G tended to exhibit higher PAI-1 level; (3) The distribution frequency of genotype 4G/4G in group C was significantly higher than in group A (42.4% vs. 28.7%, p < 0.05) and (4) In type 2 diabetic patients, the occurrence of diabetes nephropathy in genotype 4G/4G, 4G/5G and 5G/5G is 35.0%, 30.2% and 21.4%, respectively. Conclusions: (1) Plasma PAI-1 level was elevated in Type 2 diabetic patients; (2) The level of plasma PAI-1 is closely related to PAI-1 gene 4G/5G polymorphism and (3) PAI-1 4G/5G polymorphism is associated with the development and progression of predominant proteinuria diabetes nephropathy.

Association of Metabolic Syndrome with Serum Adipokines in Community-Living Elderly Japanese Women: Independent Association with Plasminogen Activator-Inhibitor-1.

Associations between metabolic syndrome (MetS) with serum adipokines and basal lipoprotein lipase mass (serum LPL) have not been extensively studied in elderly Asians, who in general have lower body mass index than European populations. A cross-sectional analysis was conducted including 159 community-living elderly Japanese women whose age averaged 77 years. MetS was defined by the modified National Cholesterol Education Program Adult Treatment Panel III criteria, but using a body mass index ≥25 kg/m(2) instead of waist circumference. Serum LPL, leptin, adiponectin, plasminogen activator inhibitor 1 (PAI-1), interleukin-6, tumor necrosis factor-alpha, and high-sensitivity C-reactive protein were measured. Both the presence of MetS and the number of MetS components were associated with higher homeostasis assessment of insulin resistance, serum levels of leptin, PAI-1, and tumor necrosis factor-alpha and with lower serum levels of LPL and adiponectin (all P < 0.05), but not with high-sensitivity C-reactive protein and interleukin-6. Among six biomarkers of MetS, PAI-1 remained associated with MetS independent of fat mass index and insulin resistance. Although proinflammatory, prothrombotic, and anti-inflammatory states were associated with MetS, higher PAI-1 was associated with MetS independent of fat mass index and insulin resistance in elderly Japanese women, in whom obesity is rare.

PAI-1 modulates cell migration in a LRP1-dependent manner via β-catenin and ERK1/2.

Plasminogen activator inhibitor-1 (PAI-1) is the major and most specific acting urokinase (uPA) and tissue plasminogen activator (tPA) inhibitor. Apart from its function in the fibrinolytic system, PAI-1 was also found to contribute to processes like tissue remodelling, angiogenesis, and tumour progression. However, the role of PAI-1 in those processes remains largely controversial with respect to the influence of PAI-1 on cell signalling pathways. Although PAI-1 does not possess its own cellular receptor, it can be bound to low-density lipoprotein receptor-related protein 1 (LRP1) which was proposed to modulate the β-catenin pathway. Therefore, we used wild-type mouse embryonic fibroblasts (MEFs), and MEFs deficient of LRP1 to study PAI-1 as modulator of the β-catenin pathway. We found that PAI-1 influences MEF proliferation and motility in a LRP1-dependent manner and that β-catenin is important for that response. In addition, expression of β-catenin and β-catenin-dependent transcriptional activity were induced by PAI-1 in wild type MEFs, but not in LRP1-deficient cells. Moreover, PAI-1-induced ERK1/2 activation was more prominent in the LRP1-deficient cells and interestingly knockdown of β-catenin abolished this effect. Together, the data of the current study show that PAI-1 can promote cell migration via LRP1-dependent activation of the β-catenin and ERK1/2 MAPK pathway which may be important in stage-specific treatment of human diseases associated with high PAI-1 levels.

Effects of urokinase type plasminogen activator and plasminogen activator inhibitor-1 expressions on the formation of aneurysm of perimembranous ventricular septal defect

The exact mechanisms of defect closure in patients with perimembranous ventricular septal defect (PMVSD) remain unknown. We hypothesized that the expression of urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) may mediate extracellular matrix (ECM) remodeling in aneurysms. Seven normal heart tricuspid septal leaflet and 33 aneurysms were collected in Shanghai Renji Hospital and Shanghai Children's Medical Center from January 2008 to June 2010. Immunohistochemical expression of uPA and PAI-1 in 4 normal heart valvular tissues and 15 aneurysms was detected with immunohistochemical methods. The expression of uPA and PAI-1 mRNA in 3 normal heart valvular tissues and 7 aneurysms was studied by real time fluorescent PCR; the protein expression of uPA and PAI-1 in 4 normal heart valvular tissues and 11 aneurysms was tested with Western blotting. The surface of the aneurysms were completely covered by endothelial cells. Two types of granulation tissue, myxoid and fibrous, were associated with the aneurismal formation. uPA were recognized predominantly in valvar interstitial cells (VICs) which located mainly in regions adjacent to the endothelium and smooth muscle cells of blood vessels. PAI-1 was found in both VICs which located mainly in granulation tissue and endothelial cells. Nine aneurysms expressed a higher uPA activity than 4 normal valvular tissues ((74.6±11.8)% vs. (49.5±7.4)%; t = 3.87, P = 0.003) and six aneurysms expressed a low uPA activity ((10.3±3.1)% vs. (49.5±7.4)%; t=11.78, P=0.000) and a high PAI-1 activity ((55.2±1.7)% vs. (50.8±3.8)%; t=2.55, P=0.034) using immunohistochemical methods. uPA / PAI-1 ratio of protein expression tested by Western blot was 0.88±0.22 in four normal heart vavular tissues; five aneurysms expressed high uPA activity and low PAI-1 activity and uPA/PAI-1 ratio was 4.26±2.04; while the other 6 cases expressed low uPA activity and high PAI-1 activity and uPA/PAI-1 ratio was 0.30±0.07; the difference among the three groups was statistically significant (P<0.05). The rate of uPA/PAI-1 in relative copy of mRNA expression among normal heart valvular tissue, high uPA expressed aneurysms and low uPA expressed aneurysms are also significantly different (2.14±0.17 vs. 0.45±0.04; 2.14±0.17 vs. 4.38±1.41, P<0.05) respectively. The expression of uPA and PAI-1 in VICs suggests that interactions among these molecules contribute to the aneurysm formation and development. This provides a potential mechanism for defect closure in patients with PMVSD.

Potential clinical relevance of uPA and PAI-1 levels in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up.

We evaluated urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) prognostic value in postmenopausal, node-negative breast cancer patients bearing tumors with estrogen receptor (ER)/progesterone receptor (PR) expression, treated with locoregional therapy alone, within an early follow-up. We focused our analysis on tumors of histological grade II in order to improve its prognostic value and, consequently, to improve a decision-making process. The cytosol extracts of 73 tumor samples were used for assessing several biomarkers. ER and PR levels were measured by classical biochemical method. Cathepsin D was assayed by a radiometric immunoassay while both uPA and PAI-1 level determinations were performed by enzyme-linked immunosorbent assays. HER-2 gene amplification was determined by chromogenic in situ hybridization (CISH) in primary tumor tissue. Patients bearing tumors smaller than or equal to 2 cm (pT1) or those with low PAI-1 levels (PAI-1 < 6.35 pg/mg) showed favorable outcome compared to patients bearing tumors greater than 2 cm (pT2,3) or those with high PAI-1 levels, respectively. Analyses of 4 phenotypes, defined by tumor size and PAI-1 status, revealed that patients bearing either pT1 tumors, irrespective of PAI-1 levels, or pT2,3 tumors with low PAI-1 levels, had similar disease-free interval probabilities and showed favorable outcome compared to those bearing pT2,3 tumors with high PAI-1 levels. Our findings suggest that tumor size and PAI-1, used in combination as phenotypes are not only prognostic but might also be predictive in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up period.

Altered plasma fibrin clot properties in essential thrombocythemia

Patients with increased thromboembolic risk tend to form denser fibrin clots which are relatively resistant to lysis. We sought to investigate whether essential thrombocythemia (ET) is associated with altered fibrin clot properties in plasma. Ex vivo plasma fibrin clot permeability coefficient (Ks), turbidimetry and clot lysis time (CLT) were measured in 43 consecutive patients with ET (platelet count from 245 to 991 × 103/µL) and 50 control subjects matched for age, sex and comorbidities. Fibrinolysis proteins and inhibitors together with platelet activation markers were determined. Reduced Ks (−38%, p < 0.0001) and prolonged CLT (+34%, p < 0.0001) were observed in ET. The differences remained significant after adjustment for fibrinogen and platelet count. ET was associated with a slightly shorter lag phase (−5%, p = 0.01) and higher maximum absorbency of the turbidimetric curve (+6%, p < 0.001). The ET patients had higher plasma P-selectin by 193% (p < 0.00001) and platelet factor 4 (PF4) by 173% (p < 0.00001), with higher P-selectin observed in 19 (44%) patients with JAK-2 gene V617F mutation. Higher t-PA (+20%, p < 0.001), 23% higher plasminogen activator inhibitor-1, PAI-1 (+23%, p < 0.01) and unaltered thrombin-activatable fibrinolysis inhibitor, plasminogen and α2-antiplasmin activity were found in the ET group. Ks inversely correlated with fibrinogen, PF4 and C-reactive protein. CLT positively correlated only with PAI-1. Patients with ET display prothrombotic plasma fibrin clot phenotype including impaired fibrinolysis, which represents a new prothrombotic mechanism in this disease.

Markers of inflammation and fibrinolysis in relation to outcome after surgery for lumbar disc herniation. A prospective study on 177 patients

The role of inflammation and fibrinolysis for the development of back pain and sciatica has been discussed. The aim of this study was to assess the relationship between markers of inflammation and fibrinolysis, to predict the outcome after surgery for lumbar disc herniation. 177 patients were recruited. High sensitive C-reactive protein (hsCRP), plasminogen activator inhibitor 1 (PAI-1), fibrinogen, and D-dimer were analyzed preoperatively. Visual analogue scale (VAS) for back and leg pain, Oswestry Disability Index (ODI), and EuroQol 5 Dimensions (EQ-5D) were assessed preoperatively and at 6 weeks, 6-, 12-, and 24- months postoperatively. Dichotomization was made at the median for the laboratory analyses, and between the worst quartile and the other three quartiles for the outcome variables. Logistic regression was used to determine the odds ratios (OR) and 95 % confidence intervals (CI). The associations between PAI-1 and outcome seemed to be most prominent at the 6 and 12-month follow-up. When being in the upper half of PAI-1, the OR for being in the worst quartile of VAS back pain 12 months postoperatively was 3.33 (1.56-7.10). The corresponding OR for VAS leg pain was 2.46 (1.18-5.10), for ODI 2.83 (1.35-5.94) and for EQ-5D 2.73 (1.30-5.75). The OR for hsCRP was 2.10 (1.03-4.29) for being in the worst quartile of VAS back pain. Fibrinogen or D-dimer was not associated with any outcome variable. High PAI-1, a marker of fibrinolysis, was fairly consistently associated with poor outcome, while hsCRP, fibrinogen, and D-dimer were not.

Association among Fibrinolytic Proteins, Metabolic Syndrome Components, Insulin Secretion, and Resistance in Schoolchildren.

We investigated the role of urokinase plasminogen activator (uPA) and its soluble receptors (suPAR) and plasminogen activator inhibitor-1 (PAI-1) in metabolic syndrome (MetS) components, insulin secretion, and resistance in schoolchildren. We enrolled 387 children, aged 10.3 ± 1.5 years, in Taipei. Anthropometry, fibrinolytic proteins, MetS components, insulin secretion, and resistance were measured. Subjects were divided into normal, overweight, and obese groups. Finally, the relationship between fibrinolytic proteins and metabolic syndrome in boys and girls was analyzed. In boys, PAI-1 was positively associated with body mass index (BMI) percentile, hypertriglyceride, insulin secretion, and resistance. In girls, PAI-1 was positively associated with obesity, hypertriglyceridemia, and insulin secretion. In girls, uPA was positively associated with insulin secretion. suPAR was positively associated with high-sensitivity C-reactive protein in both boys and girls, and with BMI percentile and body fat in girls. The obese boys had higher suPAR and PAI-1 levels than the normal group. The obese girls had higher uPA, suPAR, and PAI-1 than the normal group. Boys and girls with MetS had higher PAI-1. Fibrinolytic proteins, especially PAI-1, are associated with MetS components and insulin secretion in children. Fibrinolytic proteins changes were more likely to occur in girls than in boys.

Peritoneal Effluent MMP-2 and PAI-1 in Encapsulating Peritoneal Sclerosis

Recently, the use of effluent matrix metalloproteinase 2 (MMP-2) and plasminogen activator inhibitor 1 (PAI-1) as potential biomarkers of peritoneal fibrosis has been demonstrated during longitudinal follow-up of incident peritoneal dialysis (PD) patients. This study focuses on effluent MMP-2 and PAI-1 as early diagnostic markers in the preceding years of patients who develop encapsulating peritoneal sclerosis (EPS). Diagnostic test study. PD patients who developed EPS were compared with controls using a 1:3 case-control design with a minimum PD duration of 57 months. Dialysate appearance rates of MMP-2 and PAI-1. EPS cases identified by 2 experienced nephrologists and a radiologist based on predefined criteria. 11 patients developed EPS within our center. The time course of MMP-2 appearance rates, studied by means of a linear repeated-measures model 4 years prior to the diagnosis of EPS, showed no difference between long-term controls and patients with EPS. In contrast, higher PAI-1 appearance rates were found in patients with EPS compared with controls (P=0.01). At a lag time of 1 year prior to EPS diagnosis, time-specific receiver operating characteristic curve analyses indicated a discriminative ability for PAI-1 appearance rate of 0.77 (95% CI, 0.63-0.91). A discriminative capacity was absent for those of MMP-2. Low event rate of EPS prevented independent validation in this single-center study. Elevated levels of PAI-1 appearance rates are present in patients who develop EPS, pointing to progressive peritoneal fibrosis and sclerosis. The PAI-1 appearance rate has fair discriminative capacity from 3 years prior to EPS diagnosis. Therefore, effluent PAI-1 may aid in monitoring peritoneal fibrosis and serve as a biomarker for EPS.

Circulating acute phase proteins in relation to extent and composition of coronary atherosclerosis and cardiovascular outcome: Results from the ATHEROREMO-IVUS study

IntroductionWe examined whether the acute phase proteins (APPs): Alpha-1-Antitrypsin, Alpha-2-Macroglobulin, Complement C3, ferritin, haptoglobin, and Plasminogen Activator Inhibitor 1 (PAI-1) are associated with cardiovascular outcome, as well as with the extent and composition of coronary atherosclerosis as determined by intravascular ultrasound (IVUS) virtual histology (VH). MethodsIn 2008–2011, IVUS(-VH) imaging of a non-culprit coronary artery was performed in 581 patients from the ATHEROREMO-IVUS study undergoing coronary angiography for acute coronary syndrome (ACS) (n=318) or stable angina pectoris (SAP) (n=263). Coronary atherosclerotic plaque volume, composition (fibrous, fibro-fatty, dense calcium and necrotic core) and vulnerability (VH-derived thin-cap fibroatheroma (TCFA) lesions) were assessed. Major adverse cardiac events (MACE; all-cause mortality, ACS or unplanned coronary revascularization) were assessed during 1-year follow-up. We applied linear, logistic and Cox regression. ResultsMean age was 61.5±11.4years and 75.4% were men. Higher ferritin was associated with higher coronary plaque volume (beta [95% CI]: 0.19 [0.07–0.31] percent atheroma volume), for the highest vs the lowest tertile of ferritin; p for linear association=0.013. Higher PAI-1 was associated with higher rates of all-cause mortality or ACS (hazard ratio [95% CI]: 2.98 [1.10–8.06]), for the highest vs the lowest tertile of PAI-1. No clear-cut associations could be demonstrated between APPs and composition of atherosclerosis or plaque vulnerability. ConclusionsHigher circulating ferritin was associated with higher coronary plaque volume, and higher PAI-1 was associated with higher incidence of all-cause mortality or ACS. None of the APPs displayed consistent associations with composition of atherosclerosis or plaque vulnerability.

Abstract T P75: Temporal Profiles of Plasminogen Activator Inhibitor-1 Concentration and Activity Changes in Circulation after Focal Stroke and Tissue Type Plasminogen Activator Administration in Rats

Aims: Plasminogen activator inhibitor-1 (PAI-1) is the main and potent endogenous tissue-type plasminogen activator (tPA) inhibitor, but an important question on whether PAI-1 in blood stream responds and interferes with the exogenously administered tPA remains unexplored. We for the first time investigated temporal profiles of PAI-1 concentration and activity in circulation after stroke and tPA administration in rats. Methods: Permanent MCAO focal stroke of rats were treated with saline or 10mg/kg tPA at 3 hours after stroke (n=10 per group). Plasma (platelet free) PAI-1 antigen and activity levels were measured by ELISA at before stroke, 3, 4.5 (1.5 hours after saline or tPA treatments) and 24 hours after stroke. Since vascular endothelial cells and platelets are two major cellular sources for PAI-1 in circulation, we measured releases of PAI-1 from cultured endothelial cells and isolated platelets after direct tPA (4 μg/ml) exposures for 60 min in vitro by ELISA (n=4 per group). Results: At 3 hours after stroke, both plasma PAI-1 antigen and activity were significantly increased (3.09±0.67, and 3.42±0.57 fold of before stroke baseline, respectively, all data are expressed as mean±SE). At 4.5 hours after stroke, intravenous tPA administration significantly further elevated PAI-1 antigen levels (5.26±1.24), while as expected that tPA neutralized most elevated PAI-1 activity (0.33±0.05). At 24 hours after stroke, PAI-1 antigen levels returned to the before baseline level, however, there was a significantly higher PAI-1 activity (2.51±0.53) in tPA treated rats. In vitro tPA exposures significantly increased PAI-1 releases into culture medium in cultured endothelial cells (1.65±0.08) and platelets (2.02±0.17). Conclution: Our experimental results suggest that tPA administration may further elevate stroke-increased blood PAI-1 concentration, but also increase PAI-1 activity at late 24 hours after stroke. The increased PAI-1 releases after tPA exposures in vitro suggest tPA may directly stimulate PAI-1 secretions from vascular walls and circulation platelets, which partially contributes to the PAI-1 elevation observed in focal stroke rats. The underlying regulation mechanisms and pathological consequence need further investigation.

Difference in fibrinolytic capacity in young patients with venous thrombosis or ischaemic stroke

Many recent studies focus on the common pathophysiological mechanisms and risk factors for arterial and venous thrombosis. We investigated the hypothesis that fibrinolytic capacity is similar in patients with ischaemic stroke and venous thromboembolism. Retrospective study of 604 consecutive patients (age <50 years) referred to systematic thrombophilia testing at a single regional centre. The thrombophilia test included clinical variables, genetic polymorphisms, biomarkers of coagulation and a global assay of fibrinolysis that tested the patient's blood fibrinolytic capacity by application of the euglobulin fraction of plasma to a preformed clot of plasminogen-rich bovine fibrin. The patients with venous thromboembolism (n = 284) were slightly younger (32.3 vs. 33.9 years; <0.01) than those with ischaemic stroke (n = 320), had a significantly higher prevalence of obesity (28 vs. 18%; P < 0.01), higher plasminogen activator inhibitor 1 (PAI-1) activity (12.3 vs. 11.1 IU/ml; P = 0.049) and a significantly lower fibrinolytic capacity (3.4 vs. 3.9 IU/ml; P < 0.01). The lower fibrinolytic capacity in patients with venous thromboembolism was also observed in the subgroup of patients with PAI-1 activity within the normal range (n = 430, 3.7 vs. 4.1IU/ml; P < 0.01). After adjustment for age, BMI, fibrinogen concentration, PAI-1 activity and tissue plasminogen activator activity, fibrinolytic capacity still differed significantly between the two groups. Our results indicate that the capacity for fibrinolysis is lower in young patients with venous thromboembolism than ischaemic stroke, suggesting a different mechanistic role of fibrinolysis in arterial and venous thromboembolism.

Enhancing the Function of CD34+ Cells by Targeting Plasminogen Activator Inhibitor-1

Previously, we showed that transient inhibition of TGF- β1 resulted in correction of key aspects of diabetes-induced CD34+ cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1), a major gene target of TGF-β1 activation. Using gene array studies, we examined CD34+ cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34+ cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- β1 phosphorodiamidate morpholino oligo (PMO) reduced PAI-1 mRNA in diabetic (p<0.01) and non-diabetic (p=0.05) CD34+ cells. To reduce PAI-1 in human CD34+ cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34+ cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34+ cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.

Mental Disorders and Thrombotic Risk

Patients with psychosis, severe depression, or chronic stress are at increased risk for thromboembolism. Evidence suggests that tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) imbalance may play an important role in pathophysiology of mental and thromboembolic disorders. tPA facilitates clot dissolution and participates in several brain functions, including response to stress, learning, and memory. Depression is characterized by high PAI-1 levels, and conditions related to a hypofibrinolytic status, such as polycystic ovary syndrome and metabolic syndrome, are associated with an increased risk for both depression and cardiovascular events. Depression/psychosis may occur when estrogen and progesterone levels decrease. Estrogen inhibits synthesis of transforming growth factor-β (TGF-β), a protein increasing PAI-1 synthesis, whereas progesterone induces brain-derived neurotrophic factor release, stimulating tPA expression in neurons. Synthetic progestogens (progestins) may cause severe depression. Notably, progestin metabolites, which may prevent synthesis of natural progesterone by feedback, do not cross the blood-brain barrier. Intense suprapubic menstrual cramp, which may relate to difficulty to dissolve clots, correlates with severe premenstrual tension. Exercise, which increases tPA synthesis, has antidepressive and antithrombotic effects. We suggest that thromboembolism and some mental disorders are mechanistically related: tPA-PAI-1 imbalance seems to be a common denominator.

Ten-year analysis of the prospective multicentre Chemo-N0 trial validates American Society of Clinical Oncology (ASCO)-recommended biomarkers uPA and PAI-1 for therapy decision making in node-negative breast cancer patients

AimFinal 10-year analysis of the prospective randomised Chemo-N0 trial is presented. Based on the Chemo-N0 interim results and an European Organisation for Research and Treatment of Cancer (EORTC) pooled analysis (n=8377), American Society of Clinical Oncology (ASCO) and Arbeitsgemeinschaft Gynäkologische Onkologie (AGO) guidelines recommend invasion and metastasis markers urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitor-1 (PAI-1) for risk assessment and treatment decision in node-negative (N0) breast cancer (BC). MethodsThe final Chemo-N0 trial analysis (recruitment 1993–1998; n=647; 12 centres) comprises 113 (5-167) months of median follow-up. Patients with low-uPA and PAI-1 tumour tissue levels (n=283) were observed. External quality assurance guaranteed uPA/PAI-1 enzyme-linked immunosorbent assay (ELISA) standardisation. Of 364 high uPA and/or PAI-1 patients, 242 agreed to randomisation for CMF chemotherapy (n=117) versus observation (n=125). ResultsActuarial 10-year recurrence rate (without any adjuvant systemic therapy) for high-uPA/PAI-1 observation group patients (randomised and non-randomised) was 23.0%, in contrast to only 12.9% for low-uPA/PAI-1 patients (plog-rank=0.011). High-risk patients randomised to cyclophosphamide-methotrexate-5-fluorouracil (CMF) therapy had a 26.0% lower estimated probability of disease recurrence than those randomised for observation (intention-to-treat (ITT)-analysis: hazard ratio (HR) 0.74 (0.44–1.27); plog-rank=0.28). Per-protocol analysis demonstrated significant treatment benefit: HR 0.48 (0.26–0.88), p=0.019, disease-free survival (DFS) Cox regression, adjusted for tumour stage and grade. ConclusionsChemo-N0 is the first prospective biomarker-based therapy trial in early BC defining patients reaching good long-term DFS without adjuvant systemic therapy. Using a standardised uPA/PAI-1 ELISA, almost half of N0-patients could be spared chemotherapy, while high-risk patients benefit from adjuvant chemotherapy. These 10-year results validate the long-term prognostic impact of uPA/PAI-1 and the benefit from adjuvant chemotherapy in the high-uPA/PAI-1 group at highest level of evidence. They thus support the guideline-based routine use of uPA/PAI-1 for risk-adapted individualised therapy decisions in N0 breast cancer.