Inhibitor Of Tissue-type Plasminogen Activator Research Articles

Clinical and diagnostic significance of plasminogen activator inhibitor type 1 in the early development of coronary heart disease

Aim to determine the quantitative content of the tissue plasminogen activator inhibitor type 1 (PAI-1) and to determine its relationship with risk factors for coronary artery disease.
 Material and methods. The study included 67 male and female patients (36 men and 31 women) with coronary artery disease (CAD) and stable angina classes II-IV with the presence of hypertension. The mean age of the patients was 60.20.76 years. In all patients, clinical and anamnestic data were collected during the examination. For pain assessment, we used a verbal scale. We registered the anthropometric data and body mass index. Biochemical and instrumental methods of research were used according to the standards for diagnosing CAD. We assessed the occurrence of risk factors in patients included in the study. A complex of general clinical laboratory and instrumental tests was used, as well as the method of enzyme immunoassay ELISA, which determined the level of PAI-1, platelet aggregation activity, renin, aldosterone, cortisol and endothelin.
 Results. The group of patients demonstrated a reliable elevation of PAI-1 plasma level. Compared with practically healthy people, the level of this biomarker was 1.6 times higher in patients with coronary artery disease. A significant elevation of PAI-1 level indicates an increased risk of endothelial and hemostasiological disorders in patients, which, in turn, increases the risk of thrombogenic complications. The study revealed the correlation of PAI-1 protein in CAD patients with such risk factors as mixed anxiety-depressive disorder, smoking, hypertension, obesity, and hypercholesterolemia.

PAI-1: A Major Player in the Vascular Dysfunction in Obstructive Sleep Apnea?

Obstructive sleep apnea is a chronic and prevalent condition that is associated with endothelial dysfunction, atherosclerosis, and imposes excess overall cardiovascular risk and mortality. Despite its high prevalence and the susceptibility of CVD patients to OSA-mediated stressors, OSA is still under-recognized and untreated in cardiovascular practice. Moreover, conventional OSA treatments have yielded either controversial or disappointing results in terms of protection against CVD, prompting the need for the identification of additional mechanisms and associated adjuvant therapies. Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator (tPA) and urinary-type plasminogen activator (uPA), is a key regulator of fibrinolysis and cell migration. Indeed, elevated PAI-1 expression is associated with major cardiovascular adverse events that have been attributed to its antifibrinolytic activity. However, extensive evidence indicates that PAI-1 can induce endothelial dysfunction and atherosclerosis through complex interactions within the vasculature in an antifibrinolytic-independent matter. Elevated PAI-1 levels have been reported in OSA patients. However, the impact of PAI-1 on OSA-induced CVD has not been addressed to date. Here, we provide a comprehensive review on the mechanisms by which OSA and its most detrimental perturbation, intermittent hypoxia (IH), can enhance the transcription of PAI-1. We also propose causal pathways by which PAI-1 can promote atherosclerosis in OSA, thereby identifying PAI-1 as a potential therapeutic target in OSA-induced CVD.

Dynamics of indicators of endothelial dysfunction in children with obesity

Introduction. Constitutionally exogenous obesity (CEO) belongs to a number of significant medical and social problems of the modern world, assumes epidemic proportions and leads among alimentary-dependent pathology in children. The aim of the work was to determine changes in indicators of endothelial dysfunction (ED) in children of different age with obesity of various severity. Materials and methods. One hundred twenty six children aged of 6 to 17 years were comprehensively examined, data on changes in the serum content of ED mediators in CEOs grade 1-3 were presented by quantitative determination of nitric oxide, endothelin-1, leptin, homocysteine, intercellular adhesion molecules and vascular cell adhesion-1, tissue-type plasminogen activator inhibitor antigen, Willebrand factor and his antigen. Results. The regularities of changes in the concentrations of these compounds in the blood depending on the age of patients and the degree of obesity, which reflect the functional state of the endothelial system and can serve as criteria for the severity of ED requiring adequate and timely correction in children, have been established. Conclusion. Indicators of endothelial dysfunction can serve as criteria for its severity, their detection will allow optimizing early diagnosis and determining the amount of timely therapy.

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

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

Nursing intervention in prevention of phlebothrombosis of lower limbs after renal tumor resection

Objective To observe the effect of nursing intervention based on analytic hierarchy process in prevention of phlebothrombosis of lower limbs after renal tumor resection. Methods Clinical materials of 80 patients with renal tumor resection from October 2017 to October 2019 were analyzed retrospectively. According to the application time of nursing intervention based on analytic hierarchy process, the patients treated with routine nursing intervention from October 2017 to September 2018 were selected as control group(n=34), and the patients treated with nursing intervention based on analytic hierarchy process from October 2018 to October 2019 were selected as study group(n=46). Rehabilitation related indexes, coagulation and fibrinolysis indexes after intervention, quality of life, incidence of complications and excellent rate of nursing were compared between the two groups. Results The time to anal exhaustion, infusion time, time to first activity and hospitalization time of the study group were significantly shorter than those of the control group, and the hospitalization expense was signifiantly less than that of the control group(P<0.05). After the intervention, prothrombin time(PT), D-dimer, partial thromboplastin time(APTT), plasma tissue plasminogen activator antigen(TPA-Ag)in the study group were significantly lower than those in the control group, and the tissue-type plasminogen activator inhibitor(PAI)was significantly higher than that in the control group(P<0.05). After intervention, the scores of environment, physiology, independence, psychological state, mental support, social relationship and total score in both groups increased significantly, and the - scores of environment, physiological condition, independent state, psychological state, mental support, social relationship score and total score in the study group were significantly higher than those in the control group(P<0.05). The incidence rates of lower limbs swelling or pain, venous thrombosis of lower limbs, pulmonary embolism and other complications in the study group were significantly lower than those in the control group(P<0.05). The excellent rate of nursing in the study group was 97.83%, which was significantly higher than 67.65% in the control group(P<0.05). Conclusion Nursing intervention based on analytic hierarchy process can effectively shorten the recovery time of patients with renal tumor resection, improve the quality of life of patients, and prevent phlebothrombosis of lower limbs.

ПОКАЗНИКИ ДЕЯКИХ КОМПОНЕНТІВ СИСТЕМИ АКТИВАЦІЇ ПЛАЗМІНОГЕНУ ПРИ РАКУ СЕЧОВОГО МІХУРА

Bladder cancer (BC) continues to be a disease with a high mortality rate. Bladder cancer is the sixth for men and seventeenth for women in the incidence of malignancy worldwide. The invasion and metastasis of malignant tumors are caused by a sequence of processes, including loss of cell-cell and / or cell-matrix adhesion, proteolysis, and induction of angiogenesis. Different protease systems are involved in these processes, especially during the invasion and development of metastases. One such protease system is a plasminogen activation system or fibrinolysis system. Changes in the balance of plasminogen activation systems have been investigated in many types of malignancies, and these changes may not only indicate the functioning of this system but may also have prognostic significance. In malignancies, the components of this system are involved in the growth, invasion, and metastasis of tumors, affecting cell migration and angiogenesis. The main, but a well-studied component of the plasminogen activation system is serine proteinase – urokinase-type plasminogen activator (uPA). In contrast to uPA, tissue-type plasminogen activator (tPA) is characterized by a high affinity for fibrin and is involved in thrombolysis. Both types of plasminogen activators are synthesized in tumor tissues: tPA and uPA. The largest player among the inhibitors of fibrinolysis is the plasminogen activator inhibitor type 1 (PAI-1), involved in the pathogenesis of many cardiovascular diseases, as well as in cancer. The purpose of this study was to detect changes in the content of plasminogen activator tissue type tPA and PAI-1 in the blood plasma of patients with BC at different stages of the disease. The study involved 40 men who were verified with a diagnosis of BC. The content of tPA and PAI-1 in preoperative blood plasma was determined by enzyme immunoassay in ELISA modification. In our study, changes in the tPA and PAI-1 content of the blood plasma at different stages were identified, which can characterize tumor growth and invasion and can supplement existing disease information.

Amyloid beta soluble forms and plasminogen activation system in Alzheimer's disease: Consequences on extracellular maturation of brain-derived neurotrophic factor and therapeutic implications.

SummarySoluble oligomeric forms of amyloid beta (Aβ) play an important role in causing the cognitive deficits in Alzheimer’s disease (AD) by targeting and disrupting synaptic pathways. Thus, the present research is directed toward identifying the neuronal pathways targeted by soluble forms and, accordingly, develops alternative therapeutic strategies. The neurotrophin brain‐derived neurotrophic factor (BDNF) is synthesized as a precursor (pro‐BDNF) which is cleaved extracellularly by plasmin to release the mature form. The conversion from pro‐BDNF to BDNF is an important process that regulates neuronal activity and memory processes. Plasmin‐dependent maturation of BDNF in the brain is regulated by plasminogen activator inhibitor‐1 (PAI‐1), the natural inhibitor of tissue‐type plasminogen activator (tPA). Therefore, tPA/PAI‐1 system represents an important regulator of extracellular BDNF/pro‐BDNF ratio. In this review, we summarize the data on the components of the plasminogen activation system and on BDNF in AD. Moreover, we will hypothesize a possible pathogenic mechanism caused by soluble Aβ forms based on the effects on tPA/PAI‐1 system and on the consequence of an altered conversion from pro‐BDNF to the mature BDNF in the brain of AD patients. Translation into clinic may include a better characterization of the disease stage and future direction on therapeutic targets.

Association between smoking status assessed with plasma-cotinine and inflammatory and endothelial biomarkers in HIV-positive and HIV-negative individuals.

Smoking is a major contributor to mortality and morbidity in HIV-positive individuals. Our primary objective was to evaluate the association between smoking status determined by plasma cotinine (P-cotinine) concentration and inflammatory and endothelial biomarkers in HIV-positive versus HIV-negative individuals. We studied eight inflammatory/endothelial biomarkers [high-sensitivity C-reactive protein (hsCRP), E-selectin, soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble intercellular adhesion molecule 1 (sICAM-1), matrix metallopeptidase 9 (MMP-9), myeloperoxidase (MPO), tissue type plasminogen activator inhibitor 1 (tPAI) and endothelin] in 105 HIV-positive individuals and 105 HIV-negative individuals matched on age, sex and self-reported smoking status. Smoking status was determined using P-cotinine (a concentration > 14 ng/mL was defined as demonstrating exposure to smoke). We used linear regression models to (1) examine the association between smoking status and inflammatory/endothelial biomarkers in HIV-positive compared with HIV-negative individuals, and (2) to determine whether there was evidence to suggest that the impact of smoking status on the biomarkers differed between HIV-positive and HIV-negative individuals. Of the eight biomarkers, smokers had increased hsCRP, sICAM-1 and MMP-9 concentrations irrespective of HIV status and increasing P-cotinine concentration was associated with increasing hsCRP concentration. We found no interaction between smoking and HIV status. HIV infection was associated with increased hsCRP, E-selectin, sVCAM-1, sICAM-1 and MMP-9 concentrations. Self-reported smoking status differed substantially from smoking status assessed with P-cotinine. Several biomarkers were associated with smoking status and HIV status. However, our data do not indicate that the effects of smoking on the biomarkers differ between HIV-positive and HIV-negative individuals.

Plasminogen Activator Inhibitor-1 Reduces Tissue-Type Plasminogen Activator–Dependent Fibrinolysis and Intrahepatic Hemorrhage in Experimental Acetaminophen Overdose

Acetaminophen (APAP)-induced liver injury in mice is associated with activation of the coagulation cascade and deposition of fibrin in liver. Plasminogen activator inhibitor-1 (PAI-1) is an important physiological inhibitor of tissue-type plasminogen activator (tPA) and plays a critical role in fibrinolysis. PAI-1 expression is increased in both experimental APAP-induced liver injury and patients with acute liver failure. Prior studies have shown that PAI-1 prevents intrahepatic hemorrhage and mortality after APAP challenge, but the downstream mechanisms are not clear. We tested the hypothesis that PAI-1 limits liver-related morbidity after APAP challenge by reducing tPA-dependent fibrinolysis. Compared with APAP-challenged (300 mg/kg) wild-type mice, hepatic deposition of cross-linked fibrin was reduced, with intrahepatic congestion and hemorrhage increased in PAI-1-deficient mice 24 hours after APAP overdose. Administration of recombinant wild-type human PAI-1 reduced intrahepatic hemorrhage 24 hours after APAP challenge in PAI-1-/- mice, whereas a mutant PAI-1 lacking antiprotease function had no effect. Of interest, tPA deficiency alone did not affect APAP-induced liver damage. In contrast, fibrinolysis, intrahepatic congestion and hemorrhage, and mortality driven by PAI-1 deficiency were reduced in APAP-treated tPA-/-/PAI-1-/- double-knockout mice. The results identify PAI-1 as a critical regulator of intrahepatic fibrinolysis in experimental liver injury. Moreover, the results suggest that the balance between PAI-1 and tPA activity is an important determinant of liver pathology after APAP overdose.

Plasminogen activator inhibitor-1 as a predictor after cardiopulmonary bypass for postoperative atrial fibrillation

Background Following cardiac surgery, atrial fibrillation may be a common event. While postoperative atrial fibrillation (POAF) can be transient, it may lead to serious consequences, such as stroke, hemodynamic instability, and death. Plasminogen activator inhibitor-1 (PAI-1) serves as the primary inhibitor of tissue-type plasminogen activator, but also is mainly an acute-phase reactant. Increased PAI-1 promotes fibrosis and reduces extracellular matrix turnover, which modify the atrial substrate and potentially lead to POAF trigged by cardiac surgery. Aim The aim the study was to assess the efficacy of PAI-1 as a predictor of POAF after cardiopulmonary bypass (CPB). Patients and methods In this study, we enrolled 100 patients undergoing cardiac surgery requiring CPB and were in sinus rhythm at surgery time. Blood samples were obtained for the measurement of PAI-1 in the morning of the operation and immediately after separation from CPB and administration of protamine. Pearson’s χ2-test, Fisher’s exact test, area under the receiver operating characteristic curves, P-value less than 0.05, multivariable binary logistic regression were used. Results This study has shown that the serum level of preoperative PAI-1 more than 15 ng/ml and post-CPB level of PAI-1 more than 23 ng/ml are associated with high incidence of POAF (P 60 years) (P=0.04), hypertension history (P=0.035), number of grafts (P=0.01), Right Coronary Artery (RCA) graft (P Conclusion PAI-1 could be considered as a predictor of POAF whether measured preoperatively or postoperatively immediately after separation from CPB.

A hydrophobic patch surrounding Trp154 in human neuroserpin controls the helix F dynamics with implications in inhibition and aggregation

Neuroserpin (NS) mediated inhibition of tissue-type plasminogen activator (tPA) is important for brain development, synapse formation and memory. Aberrations in helix F and β-sheet A movement during inhibition can directly lead to epilepsy or dementia. Conserved W154 residue in a hydrophobic patch between helix F and β-sheet A is ideally placed to control their movement during inhibition. Molecular Dynamics (MD) simulation on wild type (WT) NS and its two variants (W154A and W154P) demonstrated partial deformation in helix F and conformational differences in strands 1A and 2A only in W154P. A fluorescence and Circular Dichroism (CD) analysis with purified W154 variants revealed a significant red-shift and an increase in α-helical content in W154P as compared to W154A and WT NS. Kinetics of tPA inhibition showed a decline in association rates (ka) for W154A as compared to WT NS with indication of complex formation. Appearance of cleaved without complex formation in W154P indicates that the variant acts as substrate due to conformational misfolding around helix F. Both the variants however showed increased rate of aggregation as compared to WT NS. The hydrophobic patch identified in this study may have importance in helix F dynamics of NS.

Generation and in vitro characterisation of inhibitory nanobodies towards plasminogen activator inhibitor 1.

Plasminogen activator inhibitor 1 (PAI-1) is the principal physiological inhibitor of tissue-type plasminogen activator (t-PA) and has been identified as a risk factor in cardiovascular diseases. In order to generate nanobodies against PAI-1 to interfere with its functional properties, we constructed three nanobody libraries upon immunisation of three alpacas with three different PAI-1 variants. Three panels of nanobodies were selected against these PAI-1 variants. Evaluation of the amino acid sequence identity of the complementarity determining region-3 (CDR3) reveals 34 clusters in total. Five nanobodies (VHH-s-a98, VHH-2w-64, VHH-s-a27, VHH-s-a93 and VHH-2g-42) representing five clusters exhibit inhibition towards PAI-1 activity. VHH-s-a98 and VHH-2w-64 inhibit both glycosylated and non-glycosylated PAI-1 variants through a substrate-inducing mechanism, and bind to two different regions close to αhC and the hinge region of αhF; the profibrinolytic effect of both nanobodies was confirmed using an in vitro clot lysis assay. VHH-s-a93 may inhibit PAI-1 activity by preventing the formation of the initial PAI-1t-PA complex formation and binds to the hinge region of the reactive centre loop. Epitopes of VHH-s-a27 and VHH-2g-42 could not be deduced yet. These five nanobodies interfere with PAI-1 activity through different mechanisms and merit further evaluation for the development of future profibrinolytic therapeutics.

Cell density-dependent stimulation of PAI-1 and hyaluronan synthesis by TGF-β in orbital fibroblasts.

During the course of Graves' orbitopathy (GO), orbital fibroblasts are exposed to factors that lead to proliferation and extracellular matrix (ECM) overproduction. Increased levels of tissue plasminogen activator inhibitor type 1 (PAI-1 (SERPINE1)) might promote the accumulation of ECM components. PAI-1 expression is regulated by cell density and various cytokines and growth factors including transforming growth factorβ(TGF-β). We examined the effects of increasing cell densities and TGF-β on orbital fibroblasts obtained from GO patients and controls. Responses were evaluated by the measurement of proliferation, PAI-1 expression, and ECM production. There was an inverse correlation between cell density and the per cell production of PAI-1. GO orbital, normal orbital, and dermal fibroblasts behaved similarly in this respect. Proliferation rate also declined with increasing cell densities. Hyaluronan (HA) production was constant throughout the cell densities tested in all cell lines. In both GO and normal orbital fibroblasts, but not in dermal fibroblasts, TGF-β stimulated PAI-1 production in a cell density-dependent manner, reaching up to a five-fold increase above baseline. This has been accompanied by increased HA secretion and pericellular HA levels at high cell densities. Increasing cell density is a negative regulator of proliferation and PAI-1 secretion both in normal and GO orbital fibroblasts; these negative regulatory effects are partially reversed in the presence of TGF-β. Cell density-dependent regulation of PAI-1 expression in the orbit, together with the local cytokine environment, may have a regulatory role in the turnover of the orbital ECM and may contribute to the expansion of orbital soft tissue in GO.

Structural basis of specific inhibition of tissue-type plasminogen activator by plasminogen activators inhibitor-1.

Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1) [4] (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) [19]; (2) formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3) solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19], [20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19].

Effective tranexamic acid concentration for 95% inhibition of tissue-type plasminogen activator induced hyperfibrinolysis in children with congenital heart disease: A prospective, controlled, in-vitro study.

Although recent studies have assessed tranexamic acid (TXA) pharmacokinetics in different subgroups, the effective concentration of TXA required to completely inhibit fibrinolysis remains to be determined. An in-vitro determination of the effective TXA concentration needed for 95% inhibition (EC95) of tissue-type plasminogen activator (t-PA) activated fibrinolysis, using an experimental model designed for thromboelastometry (ROTEM). A prospective interventional study. Department of Anaesthesiology, Queen Fabiola Children's University Hospital and Laboratory of Haematology and Haemostasis, Brugmann University Hospital. Patients were enrolled between June 2013 and October 2014. Twenty children, aged between 1 and 10 years, undergoing elective cardiac catheterisation were included (10 with cyanotic and 10 with noncyanotic diseases). Exclusion criteria were child requiring a procedure in a moribund state. Ten adult volunteers were also included as controls. Citrated whole blood samples were obtained from children and volunteers. The extrinsic coagulation pathway was activated by tissue factor using the EXTEM test on ROTEM. The degree of lysis measured 30 min (LI30) after the clotting time (CT), and clot amplitudes measured at different times were recorded at baseline, after addition of 1535 units t-PA ml(-1), and following the addition of increasing TXA concentrations in t-PA activated samples. The concentration-effect analysis performed with lysis index after 30 min (LI30) allowed the determination of TXA efficacy concentration 50% (EC50), and calculation of the EC95, which was significantly lower in cardiac surgery children than in adults [8.6 μg ml(-1); 95% confidence interval (95% CI) 6.9 to 14.9 versus 11.3 μg ml(-1); 95% CI 10.6 to 12.9, P < 0.001]. In this in-vitro study, we observed that the EC95 TXA concentration that completely inhibited t-PA induced hyperfibrinolysis in children with congenital heart was significantly lower than the concentration required in healthy adult volunteers. Further studies are needed to confirm that this plasma concentration can effectively inhibit fibrinolysis activation in children undergoing cardiac surgery.

Abstract 17261: Inhibition of Fibrinolysis Attenuates Influenza A-induced Lung Injury

Rationale: Influenza A virus (IAV) is the leading cause of death from an infectious cause and ranks 8th in the list of attributable annual mortality in the United States. Currently available antiviral treatments are limited and may become ineffective as resistant strains emerge. The coexistence of hemostatic alterations with inflammatory responses to viral infections support the idea that common molecular mechanisms contribute to the regulation of these systems. Previous studies demonstrated that IAV induces the activation of both coagulation and fibrinolytic pathways, and that the impairment of the latter is mainly attributable to high levels of plasminogen activator inhibitor type I (PAI-1), the major inhibitor of tissue-type plasminogen activator (t-PA). Interferon-α (IFN-α) plays a key role in the control of viral replication and overall shaping of the immune response. We sought to determine whether PAI-1, plays a role in the immune response and morbidity and mortality associated with IAV infection. Methods: Wild-type (C57BL/6J), PAI-1-/-, t-PA-/- and PAI-1 stab (transgenic mice expressing an active form of human PAI-1) mice were treated intratracheally with IAV (A/WSN/33 [H1N1] 500 pfu/mouse in 50μl PBS). We assessed the levels of IFN-α in bronchoalveolar lavage fluid (BALF) on day 2 and 4, and influenza A-induced morbidity (weight loss) and mortality. Results: Compared with wild-type mice, PAI-1-/- mice had increased IAV-induced morbidity and mortality. In contrast, both PAI-1 stab and t-PA-/- mice had improved survival. In wild-type mice, IAV induced a significant elevation IFN-α levels in BALF. PAI-1 stab mice had a 2-fold increase, while PAI-1-/-mice had a 69% reduction in IFN-α levels in BALF compared to wild-type mice. Conclusions: Inhibition of fibrinolysis by means of genetic overexpression of PAI-1 or deletion of t-PA augments the antiviral response and attenuates the morbidity and mortality associated with IAV infection. These results suggest an important functional role for PAI-1 and fibrinolysis in the pathogenesis of influenza A infection. Modulation of the fibrinolytic pathway or PAI-1 may potentially be useful as a target for novel therapeutics in the management of IAV-induced lung injury.

A novel polymorphism in the PAI-1 gene promoter enhances gene expression. A novel pro-thrombotic risk factor?

IntroductionPlasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of tissue-type plasminogen activator in plasma and the most important regulator of the fibrinolytic pathway. The 4G/5G polymorphism (rs1799889) in the PAI-1 promoter is associated with altered PAI-1 transcription. We have identified a new 4G/5G allele, in which a T is inserted near the 4G tract or replaces a G in the 5G tract, forming a T plus 4G (T4G) region. Materials and MethodsThis new variant was first identified in two women, one had experienced juvenile myocardial infarction, the other repeated miscarriage; both had increased PAI-1 plasma activity. In view of the important influence of this promoter region on PAI-1 protein plasma level, we performed in vitro evaluation of the effects of the T4G variant on the transcription activity of the PAI-1 gene promoter. Results and ConclusionsIn silico prediction analysis showed that presence of the T4G allele disrupts the E-Box region upstream of the T4G variant, altering the affinity of the target sequence for E-Box binding factors like upstream stimulatory factor-1 (USF-1). Basal T4G promoter activity was 50% higher compared to 4G and 5G variants, but it was less stimulated by USF-1 overexpression. We also analyzed the effects of IL-1β and IL-6 on the PAI-1 promoter activity of our three constructs and showed that the T4G variant was less affected by IL-1β than the other variants. These findings indicate that the T4G variant may be a novel risk factor for thrombotic events.

Circadian Variability of Fibrinolytic Markers and Endothelial Function in Patients with Obstructive Sleep Apnea

Obstructive sleep apnea (OSA) is strongly associated with cardiovascular disease, including stroke and acute coronary syndromes. Plasminogen activator inhibitor-1 (PAI-1), the principal inhibitor of tissue-type plasminogen activator (t-PA), has a pronounced circadian rhythm and is elevated in both OSA and cardiovascular disease and may be an important link between the two conditions. Endothelial dysfunction is one of the underlying pathophysiological mechanisms of cardiovascular disease, and may be altered in OSA. Our primary aim was to compare circadian variability of PAI-1 and t-PA in patients with OSA and normal controls by determining the amplitude (peak level) and mesor (rhythm adjusted mean) of PAI-1 and t-PA in serial blood samples over a 24-h period. The secondary aim was to measure markers of endothelial function (brachial and radial artery flow) in patients with OSA compared with normal controls. Cross-sectional cohort study. Subjects age 18 y or older, with a body mass index of 25-45 kg/m(2), with or without evidence of untreated OSA. Plasma samples were collected every 2 h, in OSA patients and matched controls, over a 24-h period. PAI-1 and t-PA antigen and activity were measured. The presence or absence of OSA (apnea-hypopnea index of 5 or greater) was confirmed by overnight polysomnography. Endothelial function was measured via brachial artery flow mediated vasodilatation and computerized arterial pulse waveform analysis. The rhythm-adjusted mean levels of PAI-1 antigen levels in the OSA group (21.8 ng/mL, 95% confidence level [CI], 18 to 25.7) were significantly higher as compared to the non-OSA group (16 ng/mL, 95% CI, 12.2 to 19.8; P = 0.03). The rhythm-adjusted mean levels of PAI-1 activity levels in the OSA group (23.9 IU/mL, 95% CI, 21.4 to 26.5) were also significantly higher than in the non-OSA group (17.2 IU/ mL, 95% CI, 14.6 to 19.9; P < 0.001).There were strong correlations between amplitude of PAI-1 activity and severity of OSA as measured by AHI (P = 0.02), and minimum oxygen levels during sleep (P = 0.04). Endothelial function parameters did not differ significantly between the two groups. The presence of obstructive sleep apnea adversely affects circadian fibrinolytic balance with higher mean plasminogen activator inhibitor-1 activity and antigen, and significantly lower mean tissue-type plasminogen activator activity compared with controls. This perturbation may be an important mechanism for increased cardiovascular events in patients with obstructive sleep apnea. Intermittent hypoxia and changes in circadian clock gene activity in obstructive sleep apnea may be responsible for these findings and warrant further study. Favorable changes in fibrinolytic balance may underlie the reduction in cardiovascular events observed with the treatment of obstructive sleep apnea.

Increased prevalence of coronary artery disease risk markers in patients with chronic hepatitis C &amp;ndash; a cross-sectional study

ObjectiveChronic hepatitis C is a global health problem and has been associated with coronary artery disease. Our aim was to examine the prevalence of coronary artery disease risk markers including endothelial biomarkers in patients with chronic hepatitis C and matched comparisons without manifest cardiovascular disease or diabetes in a cross-sectional design.MethodsSixty patients with chronic hepatitis C (mean age 51 years) were recruited from the Department of Infectious Diseases at Copenhagen University Hospital, and compared with 60 age-matched non-hepatitis C virus-infected individuals from a general population survey. We examined traditional coronary artery disease risk factors, metabolic syndrome, carotid intima media thickness, and a range of endothelial biomarkers.ResultsPatients with chronic hepatitis C had more hypertension (40% versus 25%, prevalence ratio [PR] 1.6; 95% confidence interval [CI] 0.9–2.7) and smoked more (53% versus 38%, PR 1.4; 95% CI 0.9–2.1). The two groups had similar body mass index (mean 25.0 versus 25.7 kg/m2), whereas those with chronic hepatitis C had less dyslipidemia (including significantly lower low-density lipoprotein and cholesterol/high-density lipoprotein ratio), higher glycosylated hemoglobin level (mean 6.2 versus 5.7, difference of means 0.5; 95% CI 0.3–0.8), and a higher prevalence of metabolic syndrome (28% versus 18%, PR 1.6; 95% CI 0.8–3.0). Increased carotid intima media thickness above the standard 75th percentile was seen more frequently in chronic hepatitis C (9% versus 3%, PR 1.7; 95% CI 0.4–6.7), though difference of means was only 0.04 mm (95% CI 0.00–0.10). Patients with chronic hepatitis C had increased hsCRP (high-sensitivity C-reactive protein), sICAM-1 (soluble intercellular adhesion molecule-1), sVCAM-1 (soluble vascular cell adhesion molecule-1), and soluble E-selectin, but lower levels of tPAI-1 (tissue-type plasminogen activator inhibitor-1), MMP9 (matrix metallopeptidase 9), and MPO (myeloperoxidase) than their comparisons.ConclusionOur findings indicate that patients with chronic hepatitis C have increased prevalence of several coronary artery disease risk markers. These results may be important when evaluating the appropriateness of screening for coronary artery disease and its risk factors in chronic hepatitis C.

HSP47 regulates ECM accumulation in renal proximal tubular cells induced by TGF-β1through ERK1/2 and JNK MAPK pathways

Heat shock protein (HSP)47 is a collagen-specific molecular chaperone that is essential for the biosynthesis of collagen molecules. It is likely that increased levels of HSP47 contribute to the assembly of procollagen and thereby cause an excessive accumulation of collagens in disease processes associated with fibrosis. Although HSP47 promotes renal fibrosis, the underlying mechanism and associated signaling events have not been clearly delineated. We examined the role of HSP47 in renal fibrosis using a rat unilateral ureteral obstruction model and transforming growth factor (TGF)-β(1)-treated human proximal tubular epithelial (HK-2) cells. An upregulation of HSP47 in both in vivo and in vitro models was observed, which correlated with the increased synthesis of extracellular matrix (ECM) proteins and expression of tissue-type plasminogen activator inhibitor (PAI)-1. Blockade of HSP47 by short interfering RNA suppressed the expression of ECM proteins and PAI-1. In addition, TGF-β(1)-induced HSP47 expression in HK-2 cells was attenuated by ERK1/2 and JNK MAPK inhibitors. These data suggest that ERK1/2 and JNK signaling events are involved in modulating the expression of HSP47, the chaperoning effect of which on TGF-β(1) would ultimately contribute to renal fibrosis by enhancing the synthesis and deposition of ECM proteins.