PAI-1 Antigen Research Articles

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.

Systematic review and meta-analysis of within-subject and between-subject biological variation data of coagulation and fibrinolytic measurands.

The diagnosis and monitoring of bleeding and thrombotic disorders depend on correct haemostatic measurements. The availability of high-quality biological variation (BV) data is important in this context. Many studies have reported BV data for these measurands, but results are varied. The present study aims to deliver global within-subject (CVI) and between-subject (CVG) BV estimates for haemostasis measurands by meta-analyses of eligible studies, by assessment with the Biological Variation Data Critical Appraisal Checklist (BIVAC). Relevant BV studies were graded by the BIVAC. Weighted estimates for CVI and CVG were obtained via meta-analysis of the BV data derived from BIVAC-compliant studies (graded A-C; whereby A represents optimal study design) performed in healthy adults. In 26 studies BV data were reported for 35 haemostasis measurands. For 9 measurands, only one eligible publication was identified and meta-analysis could not be performed. 74% of the publications were graded as BIVAC C. The CVI and CVG varied extensively between the haemostasis measurands. The highest estimates were observed for PAI-1 antigen (CVI 48.6%; CVG 59.8%) and activity (CVI 34.9%; CVG 90.2%), while the lowest were observed for activated protein C resistance ratio (CVI 1.5%; CVG 4.5%). This study provides updated BV estimates of CVI and CVG with 95% confidence intervals for a wide range of haemostasis measurands. These estimates can be used to form the basis for analytical performance specifications for haemostasis tests used in the diagnostic work-up required in bleeding- and thrombosis events and for risk assessment.

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.

Abstract 10876: Thrombo-Inflammatory Biomarkers in Pulmonary Embolism as Stratified in Cancer and Non-Cancer Patient Groups

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. Cancer is prevalent in PE patients and is considered an added risk factor for the adverse outcomes. Thrombo-inflammatory biomarkers profiling provide means to risk stratify PE patients. Hypothesis: This study aimed to profile several of these biomarkers in normal and PE patients and to further demonstrate any potential difference between cancer and non-cancer patients. Methods: Four hundred patients of 18 years or older age, were included through enrolment in conjunction with 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. Control plasma samples were obtained from 50 healthy individuals. All plasma samples were analyzed for D-Dimer, PAI-1 antigen, tPA, TAFI-a, vWF, CRP, troponin and IL-6 were measured by ELISA methods. Microparticles were measured using a chromogenic functional method. Individual biomarkers were compared to normal plasma and further stratified into cancer and non-cancer. Results: All the individual biomarkers exhibited up to 50-fold elevation in PE patients compared to normal groups. D-Dimer and CRP showed the most pronounced increase. When the PE group stratified into cancer and non-cancer patients, most of the biomarkers except D-Dimer and microparticles showed varying degree of modest elevation in the cancer group. vWF, IL6 and microparticles levels were found to be significantly different in the cancer group (p<0.050). Conclusions: These studies demonstrate that thrombo-inflammatory biomarkers are upregulated in PE patients. Furthermore, PE patients with cancer exhibit amplified levels of some of the markers suggesting the increased severity of pathogenesis involving thrombo-inflammatory mechanisms.

Hemostatic Responses to Multiple Bouts of Firefighting Activity: Female vs. Male Differences in a High Demand, High Performance Occupation.

While the fire service has long been a male-dominated occupation, women’s participation in this strenuous, high risk, high performance activity has increased in recent years. Firefighting induces significant cardiovascular strain, including hemostatic disruption; however, the effect of sex on hemostatic responses has not been investigated despite evidence that there are sex-related differences in hemostatic variables at rest and following exercise. Thus, we investigated hemostatic responses in age- and BMI-matched male and female firefighters who performed 3–4 evolutions of firefighting drills over a 3 h period. Venous blood samples were collected before and after the firefighting training drills and hemostatic variables were assessed. Firefighting significantly increased platelet count and factor VIII, tissue plasminogen activator (t-PA) antigen, and t-PA activity, and decreased activated partial thromboplastin time and plasminogen activator inhibitor (PAI-1) activity. Females had lower values for epinephrine-induced platelet closure time, antithrombin III, PAI-1 activity, and PAI-1 antigen. There were no interactions between sex and time for any variables assessed. In conclusion, multiple bouts of firefighting activity resulted in a procoagulatory state. Although there were sex differences for several hemostatic variables, male and female firefighters did not differ in their hemostatic response to multiple bouts of firefighting.

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.

Can laser therapy modify the secretion of the tissue-type plasminogen activator and its inhibitor in an endothelial cell culture under hyperglycemia?

Introduction : The effect of low-level laser therapy on the secretion of tissue-type plasminogen activator (t-PA) and its inhibitor (PAI-1) in an endothelial cell (EC) culture under hyperglycemia is the subject of the presented work. Hyperglycemia associated with diabetes causes vascular EC dysfunction. Low-level laser therapy is a good method to support the pharmacological treatment of diabetes complications. Materials and methods : We used lasers of 2 wavelengths: 635 nm and 830 nm with dose of 2 J/cm2. The experiment was performed in vitro in 4 groups of EC: 1 – no glucose in culture medium, no irradiation (control), 2 – glucose, no irradiation, 3 – glucose, laser 635 nm, 4 – glucose, laser 830 nm. After 2 irradiations, cells were counted and the t-PA and PAI-1 antigen (Ag) concentration in the supernatant was measured. Results : In group 2, we observed a statistically significant lower number of cells (p < 0.0001) and a higher concentration of t-PA:Ag and PAI-1:Ag (p < 0.05) compared to the control group. However, in groups 3 and 4, the number of cells increased and the concentration of t-PA:Ag and PAI-1:Ag decreased compared to group 2 and nearly reached the values in the control group. Conclusions : Hyperglycemia affects the fibrinolytic activity of ECs which is manifested by a significant increase in t-PA:Ag and PAI:Ag concentrations – recognized markers of endothelial damage. Irradiation of ECs by a low-power laser caused attenuation of the adverse effects of hyperglycemia. A tendency towards a decrease in t-PA:Ag and PAI-1:Ag concentration in the supernatant was observed with a significant increase in the number of cells to values close to control.

Regular Resistance Training Enhances Fibrinolytic Potential but Does Not Affect Coagulation.

This study aimed to identify effects of an 8-wk, whole-body RT program on coagulation and fibrinolysis. Sixteen healthy women and men (23 ± 5 yr) completed an RT program three times per week for 8 wk. Exercises included 2-3 sets of 8-12 repetitions performed at approximately 60%-80% of a one repetition maximum. Strength, body composition, and body circumferences were assessed before and after training. Plasma samples were obtained before and after training, and analyzed for active tissue plasminogen activator (tPA activity), total tissue plasminogen activator (tPA antigen), active plasminogen activator inhibitor-1 (PAI-1 activity), total plasminogen activator inhibitor-1 (PAI-1 antigen), fibrinogen, and coagulation factors VII (FVII) and VIII (FVIII). Significant increases in lean mass, arm and thigh circumferences, maximal chest press (PRE: 57.8 ± 37.5 kg, POST: 73.3 ± 43.2 kg), and leg press (PRE: 189.5 ± 96.0 kg, POST: 256.7 ± 97.9 kg) were observed (P < 0.05 for all). PAI-1 activity (PRE: 20.3 ± 32.5 IU·mL-1, POST 9.5 ± 20.9 IU·mL-1) and PAI-1 antigen decreased (PRE: 10.2 ± 9.0 ng·dL-1, POST: 7.2 ± 5.7 ng·dL-1; both, P < 0.05). No change in tPA activity or tPA antigen occurred. Fibrinogen, FVII, and FVIII did not change after training. Inhibition of fibrinolysis was decreased after training, and coagulation was unaffected. These results suggest that regular RT may beneficially influence the risk of a thrombotic event. More research is warranted to understand the mechanisms through which RT affects hemostasis.

Global Reach 2018: Influence of Excessive Erythrocytosis on Coagulation and Fibrinolytic Factors in Andean Highlanders

Increased coagulation and reduced fibrinolysis are central factors underlying thrombotic risk and events. High altitude-induced excessive erythrocytosis (EE) is prevalent in Andean highlanders, contributing to increased cardiovascular risk. Disruption in the coagulation-fibrinolytic axis resulting in uncontrolled fibrin deposition may underlie the increased thrombotic risk associated with high altitude EE. The experimental aim of this study was to determinewhether EE is associated with a prothrombotic blood coagulation and fibrinolytic profile in Andean highlanders. Plasma coagulation (von Willebrand factor [vWf], factor VII, VIII, and X) and fibrinolytic (tissue-type plasminogen activator [t-PA] and plasminogen activator inhibitor-1 [PAI-1]) factors as well as D-dimer levels were determined in 26 male residents of Cerro de Pasco, Peru (4,340 m): 12 without EE (age: 40±13 yr; Hb: 17.4±1.9 g/dL) and 14 with EE (43±15 yr; 24.4±1.6 g/dL). There were no significant differences in vWf (40.5+24.8 vs 45.5+22.4 %), factor VII (77.0+14.5 vs 72.5+8.9 %), factor VIII (55.6+19.8 vs 60.7+26.8 %) and factor X (73.9+8.3 vs 67.3+10.9 %) between the Andean highlanders without or with EE. t-PA antigen (8.5+3.6 vs 9.6+5.4 ng/mL), t-PA activity (5.5+2.4 vs 5.8+1.6 IU/mL), PAI antigen (45.0+33.8 vs 40.5+15.8 ng/mL), PAI-1 activity (0.24+0.09 vs 0.25+0.11 IU/mL) and the molar concentration ratio of active t-PA to active PAI-1 (1:0.051+0.034 vs 1:0.046+0.021 mmol/L) were also similar between the groups as were D-dimer levels (235.0+126.4 ng/mL vs 268.4+173.7 ng/mL). In conclusion, the results of the present study indicate that EE is not associated with prothrombotic disruptions in coagulation or the fibrinolytic system in Andean highlanders. Although dysregulation in both pathways is known to increase thrombotic risk, EE does not appear to directly influence either pathway to affect vascular risk in Andean men.

Global REACH 2018: Influence of excessive erythrocytosis on coagulation and fibrinolytic factors in Andean highlanders.

What is the central question of this study? Are coagulation and fibrinolytic factors disrupted in Andean highlanders with excessive erythrocytosis? What is the main finding and its importance? Excessive erythrocytosis is not associated with prothombotic disruptions in coagulation or the fibrinolytic system in Andean highlanders. Impairments in coagulation and fibrinolysis may not contribute to the increased vascular risk associated with excessive erythrocytosis. Increased coagulation and reduced fibrinolysis are central factors underlying thrombotic risk and events. High altitude-induced excessive erythrocytosis (EE) is prevalent in Andean highlanders, contributing to increased cardiovascular risk. Disruption in the coagulation-fibrinolytic axis resulting in uncontrolled fibrin deposition might underlie the increased thrombotic risk associated with high-altitude EE. The experimental aim of this study was to determine whether EE is associated with a prothrombotic blood coagulation and fibrinolytic profile in Andean highlanders. Plasma coagulation factors (von Willebrand factor and factors VII, VIII and X), fibrinolytic factors [tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1)] and D-dimer levels were determined in 26 male residents of Cerro de Pasco, Peru (4340m a.s.l.): 12 without EE (age, 40±13years; haemoglobin, 17.4±1.9g/dl) and 14 with EE (age, 43±15years; haemoglobin, 24.4±1.6g/dl). There were no significant differences in von Willebrand factor (40.5±24.8 vs. 45.5±22.4%), factorVII (77.0±14.5 vs. 72.5±8.9%), factorVIII (55.6±19.8 vs. 60.7±26.8%) and factorX (73.9±8.3 vs. 67.3±10.9%) between the Andean highlanders without or with EE. The t-PA antigen (8.5±3.6 vs. 9.6±5.4ng/ml), t-PA activity (5.5±2.4 vs. 5.8±1.6IU/ml), PAI antigen (45.0±33.8 vs. 40.5±15.8ng/ml), PAI-1 activity (0.24±0.09 vs. 0.25±0.11IU/ml) and the molar concentration ratio of active t-PA to active PAI-1 (1:0.051±0.034 vs. 1:0.046±0.021mmol/l) were also similar between the groups, as were D-dimer levels (235.0±126.4 vs. 268.4±173.7ng/ml). Collectively, the results of the present study indicate that EE is not associated with a hypercoagulable, hypofibrinolytic state in Andean highlanders.

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.

Hypofibrinolysis is associated with the severity of Covid-19 infection: The role of obesity

Introduction: Obesity, by causing hypofibrinolysis and thrombotic complications, ought to be a risk factor in terms of disease severity and mortality in COVID-19 infection.We aim to investigate the effects of obesity on fibrinolytic system in coronavirus patients while studying the changes of major fibrinolytic inhibitors plasminogen activator inhibitor (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI). Material and methods: Sixty-six patients and 21 healthy donors were investigated and observed until either their recovery or death. Body mass index (BMI) was calculated for each person and peripheral blood used for PAI-1 and TAFI measurements. PAI-1 antigen and TAFI activated/inactivated (TAFIa/i) were measured using ELISA kits. Results: PAI-1 antigen and TAFIa/i plasma levels are higher in patients than in control group (P<00001 for both).Patients needing ICU had higher TAFIa/i values than non-ICU patients. The 15 patients who died had higher TAFIa/i levels than those staying alive.Obese patients (BMI . 30) had higher PAI-1 levels than non-obese patients. TAFIa/i is associated with D-dimer, C-reactive protein (CRP), ferritin, creatinine and neutrophile count, whereas PAI-1 is associated only with the serum creatinine level. Conclusion: Our study shows that the levels of the major fibrinolytic inhibitors PAI 1 and TAFI increase in patients with COVID-19 infection, and proves for the first time that PAI 1 levels increase more in obese patients than in non-obese ones. It is also revealed that the plasma TAFI level is associated with the severity of COVID-19 infection, which suggests that the inhibitory treatments against TAFI should be effective in preventing thrombotic complications in the course of COVID-19 infection.

Hemostasis, Hematoma Expansion, and Outcomes after Intracerebral Hemorrhage

Background: Intracerebral hemorrhage (ICH) is a serious complication of stroke with significant morbidity and mortality. Intracerebral bleeding and hematoma expansion (HE) cause displacement and damage to brain tissue leading to disability and death. An important approach to improve patient outcomes from ICH is to prevent HE. Our ability to improve patient outcomes depends on our ability to recognize HE and the associated hemostatic mechanisms. This study is designed to evaluate biomarkers of platelet function, coagulation and fibrinolysis and relate these biomarkers to statistically powerful estimates of HE and patient outcomes. Study Design and Methods: Patients with acute ICH within the first 12 hours of the onset of symptoms will be studied to identify hemostasis biomarkers which may predict hematoma expansion. Major inclusion criteria: Spontaneous ICH within 12 hours of symptom onset; age at least 21 years; consent from the patient or a representative. Exclusion criteria: hereditary hemostasis disorders, history of malignancy, vascular malformation suspected or on imaging, sepsis, comorbid condition that is likely to be disabling or fatal, hemoglobin less than 7.5 g/dL, pregnancy. Statistical methods: We will test the hypothesis that each hemostasis biomarker predicts HE by using Mann-Whitney U (for non-parametric) or ANOVA (for normally distributed results). We will set P&lt;0.01 given the multiple biomarkers to be assessed. The 80% power using a Mann-Whitney U to test the hypothesis that the distributions of HE are 3 mL different would require 147 patients (calculations made with PASS v.12, NCSS Inc., Kaysville, UT). Machine learning techniques will be applied to hemostasis biomarkers to predict the modified Rankin score (mRS), T Scores of HRQoL, and T Scores of NIH Toolbox outcomes. Precision-recall and receiver operating characteristic (ROC) curves will be constructed, and area under the curve will be compared. Biomarker selection or companion diagnostics: This study will measure platelet activity, coagulation activation and fibrinolysis in patients with acute onset (less than 12 hours) intracerebral hemorrhage ICH to determine hemostatic mechanisms of hematoma expansion. Global activity of platelets, coagulation factors and fibrinolysis activity are measured by thromboelastogram (TEG) and rotational thromboelastometry (ROTEM) assays. Platelet activity is also measured with Platelet Function assay, PFA-100, Platelet Aspirin Assay and the Platelet P2Y12 Assay. Coagulation activation is measured by Tissue Factor antigen and activity and Thrombin Generation Assay. Fibrinolysis is assayed by Plasmin/antiplasmin (PAP) complex, Fibrinogen, D-Dimer and PAI-1 antigen and activity. Study treatment, endpoints: The hemostatic parameters and hematoma expansion are compared to patient outcomes measured by the mRS and health related quality of life (HRQoL) and objective outcomes measures from the NIH toolbox to determine the relationship to patient outcomes. The mRS is a standard outcome measure for patients with ICH. The NIH developed and validated HRQoL measures Patient-Reported Outcomes Measurement Information System (PROMIS), Quality of Life in Neurological Disorders (Neuro-QoL), and objective function NIH Toolbox are the most sensitive and provide statistically powerful estimates of patient outcomes in survivors of ICH. Future Directions: This study is designed to identify hemostatic factor abnormalities and mechanisms of ICH hematoma expansion treatable with targeted therapies (e.g., desmopressin to counteract aspirin effect or fibrinolysis inhibitors). Machine learning algorithms will be employed for predicting patient outcomes after ICH and may illuminate when HE is most impactful and determine specific therapies which improve patient outcomes. This study is funded by a National Institutes of Health (NIH)/ The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) grant ID: SP0049593; R01 NS110779. Disclosures Naidech: NIH: Other: NS110779-Grant.

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.

Adiponectin Inhibits TNF-α-Activated PAI-1 Expression Via the cAMP-PKA-AMPK-NF-κB Axis in Human Umbilical Vein Endothelial Cells

Background: Tumor necrosis factor (TNF)-α can upregulate the expression of plasminogen activator inhibitor (PAI)-1, an inhibitor of fibrinolysis. Adiponectin (Adp) antagonizes TNF-α by negatively regulating its expression in various tissues. In the present study, the ability of Adp to suppress TNF-α-induced PAI-1 upregulation and the underlying mechanisms were evaluated. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with TNF-α in the presence or absence of Adp, and PAI-1 mRNA and antigen expression, activated signaling pathways, and molecular mechanisms were analyzed by qRT-PCR and ELISA. Results: Adp decreased the TNF-α-induced upregulation of PAI-1 mRNA and protein expression and suppressed TNF-α-induced cAMP-PKA-AMPK inactivation. Adp also suppressed the TNF-α-induced NF-kB binding capability on the PAI-1 promoter. Moreover, these Adp-induced effects were further enhanced or prevented by treatment with the cAMP inhibitor Rp-cAMPs or activator forskolin, respectively. Conclusions: Our data suggest that Adp abrogates TNF-α-activated PAI-1 expression by activating cAMP-PKA-AMPK signaling to suppress NF-kB binding to the PAI-1 promoter in HUVECs. Given the antifibrotic effect of PAI-1 abrogation, Adp may be utilized as a novel agent in the treatment of fibrotic diseases.

Impaired plasma clot lysis and its determinants in patients with obstructive sleep apnea syndrome.

Evidence indicates that hypercoagulability and impaired fibrinolysis have been observed in patients with obstructive sleep apnea syndrome (OSAS). It is unclear which factors determine prolonged fibrin clot lysis in OSAS. One hundred and sixty-five consecutive patients suspected of OSAS underwent overnight polysomnography. Prior to polysomnography, we determined plasma clot lysis time (CLT), plasminogen activator inhibitor (PAI)-1 antigen, activated thrombin-activatable fibrinolysis inhibitor (TAFIa), plasmin, and antiplasmin. OSAS was diagnosed in 110 (66.7%) patients, including 35 (31.8%) with severe OSAS, 26 (23.6%) with moderate OSAS, and 49 (44.6%) mild OSAS. Compared with 55 (33.3%) individuals in whom OSAS was not confirmed, OSAS patients had prolonged CLT (+12.8%), associated with higher PAI-1 antigen (+18.1%) (after adjustment for age, diabetes, and body mass index; both P < 0.01) and similar levels of TAFIa, plasmin, or antiplasmin. PAI-1, TAFIa, and CLT correlated positively with apnea/hypopnea index, which reflects the severity of OSAS (R = 0.66, P < 0.001; R = 0.29, P = 0.002; R = 0.55, P = 0.001, respectively), and with other polysomnography parameters, with the most potent correlations observed for desaturation index. Regression analysis adjusted for potential confounders showed that in OSAS, CLT was independently predicted by apnea/hypopnea index (B = 0.29, P = 0.002), PAI-1 (B = 0.42, P < 0.001), and TAFIa (B = 0.81, P = 0.044), whereas both PAI-1 and TAFIa were predicted only by desaturation index (B = 0.24, P = 0.002; and B = 0.14, P = 0.001, respectively). The severity of OSAS is closely associated with hypofibrinolysis measured in a global plasma-based assay, driven largely by PAI-1. Attenuated fibrinolysis might contribute to high risk of thromboembolic events in this disease.

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.

Effects of Body Mass Index on the Lipid Profile and Biomarkers of Inflammation and a Fibrinolytic and Prothrombotic State.

Both an overweight status and obesity are associated with high cardiovascular morbidity and mortality. The aim of this study was to examine the effects of obesity on different underlying mechanisms, i.e. inflammation, fibrinolysis and a prothrombotic state, in a young high-risk population in the Mediterranean area. The study population included 237 subjects (median age: 44 years). We recorded the presence of cardiovascular risk factors and premature ischaemic heart disease and performed weight stratification using the body mass index (BMI) according to the established World Health Organization (WHO) criteria. We also measured the serum/plasma lipid, fibrinogen, D-dimer, von Willebrand factor, tissue plasminogen activator antigen (t-PA), plasminogen activator inhibitor-1 antigen (PAI-1) and high-sensitivity C-reactive protein (CRP-hs) levels in samples of peripheral blood. The subjects with premature ischaemic heart disease and hypertension had higher BMI values (p＜0.01), and the subjects with an increased weight showed an unadjusted detrimental lipid profile, with a proinflammatory, prothrombotic state and abnormal fibrinolytic parameters. According to a multivariate analysis, the HDL-cholesterol (r(2)=0.176; p＜0.001), t-PA antigen (r(2)=0.235; p＜0.001), PAI-1 antigen (r(2)=0.164; p＜0.001) and CRP-hs (r(2)=0.096; p=0.019) levels were significantly related to the weight stratification. A high BMI is a common finding in young populations at high risk of cardiovascular disease. In the current study, the patients with an increased BMI demonstrated an unhealthy lipid profile, as well as a proinflammatory and prothrombotic state and abnormal fibrinolytic parameters.

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