Plasminogen Activator Inhibitor-1 Activity Research Articles

Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits.

Elevated active plasminogen activator inhibitor-1 (PAI-1) has an adverse effect on the outcomes of intrapleural fibrinolytic therapy (IPFT) in tetracycline-induced pleural injury in rabbits. To enhance IPFT with prourokinase (scuPA), two mechanistically distinct approaches to targeting PAI-1 were tested: slowing its reaction with urokinase (uPA) and monoclonal antibody (mAb)-mediated PAI-1 inactivation. Removing positively charged residues at the "PAI-1 docking site" (179RHRGGS184→179AAAAAA184) of uPA results in a 60-fold decrease in the rate of inhibition by PAI-1. Mutant prourokinase (0.0625-0.5 mg/kg; n = 12) showed efficacy comparable to wild-type scuPA and did not change IPFT outcomes ( P > 0.05). Notably, the rate of PAI-1-independent intrapleural inactivation of mutant uPA was 2 times higher ( P < 0.05) than that of the wild-type enzyme. Trapping PAI-1 in a "molecular sandwich"-type complex with catalytically inactive two-chain urokinase with Ser195Ala substitution (S195A-tcuPA; 0.1 and 0.5 mg/kg) did not improve the efficacy of IPFT with scuPA (0.0625-0.5 mg/kg; n = 11). IPFT failed in the presence of MA-56A7C10 (0.5 mg/kg; n = 2), which forms a stable intrapleural molecular sandwich complex, allowing active PAI-1 to accumulate by blocking its transition to a latent form. In contrast, inactivation of PAI-1 by accelerating the active-to-latent transition mediated by mAb MA-33B8 (0.5 mg/kg; n = 2) improved the efficacy of IPFT with scuPA (0.25 mg/kg). Thus, under conditions of slow (4-8 h) fibrinolysis in tetracycline-induced pleural injury in rabbits, only the inactivation of PAI-1, but not a decrease in the rate of its reaction with uPA, enhances IPFT. Therefore the rate of fibrinolysis, which varies in different pathologic states, could affect the selection of PAI-1 inhibitors to enhance fibrinolytic therapy.

Conformational preludes to the latency transition in PAI-1 as determined by atomistic computer simulations and hydrogen/deuterium-exchange mass spectrometry

Both function and dysfunction of serine protease inhibitors (serpins) involve massive conformational change in their tertiary structure but the dynamics facilitating these events remain poorly understood. We have studied the dynamic preludes to conformational change in the serpin plasminogen activator inhibitor 1 (PAI-1). We report the first multi-microsecond atomistic molecular dynamics simulations of PAI-1 and compare the data with experimental hydrogen/deuterium-exchange data (HDXMS). The simulations reveal notable conformational flexibility of helices D, E and F and major fluctuations are observed in the W86-loop which occasionally leads to progressive detachment of β-strand 2 A from β-strand 3 A. An interesting correlation between Cα-RMSD values from simulations and experimental HDXMS data is observed. Helices D, E and F are known to be important for the overall stability of active PAI-1 as ligand binding in this region can accelerate or decelerate the conformational inactivation. Plasticity in this region may thus be mechanistically linked to the conformational change, possibly through facilitation of further unfolding of the hydrophobic core, as previously reported. This study provides a promising example of how computer simulations can help tether out mechanisms of serpin function and dysfunction at a spatial and temporal resolution that is far beyond the reach of any experiment.

Inhibition of PAI-1 Activity by Toddalolactone as a Mechanism for Promoting Blood Circulation and Removing Stasis by Chinese Herb Zanthoxylum nitidum var. tomentosum.

Traditional Chinese medicine has been used to treat a variety of human diseases for many centuries. Zanthoxylum nitidum var. tomentosum is used as an adjuvant to promote blood circulation and remove stasis. However, the mechanisms of improving circulation and other biological activities of Z. nitidum var. tomentosum are still unclear. Plasminogen activator inhibitor-1 (PAI-1) regulates the plasminogen activation system through inhibition of tissue-type and urokinase-type plasminogen activators (tPA and uPA). PAI-1 has been linked to fibrin deposition that evolves into organ fibrosis and atherosclerosis. In the present study, we showed that ethanol extract prepared from Z. nitidum var. tomentosum exhibited PAI-1 inhibitory activity, and identified toddalolactone as the main active component that inhibited the activity of recombinant human PAI-1 with IC50 value of 37.31 ± 3.23 μM, as determined by chromogenic assay, and the effect was further confirmed by clot lysis assay. In vitro study showed that toddalolactone inhibited the binding between PAI-1 and uPA, and therefore prevented the formation of the PAI-1/uPA complex. Intraperitoneal injection of toddalolactone in mice significantly prolonged tail bleeding and reduced arterial thrombus weight in a FeCl3-induced thrombosis model. In addition, the hydroxyproline level in the plasma and the degree of liver fibrosis in mice were decreased after intraperitoneal injection of toddalolactone in CCl4-induced mouse liver fibrosis model. Taken together, PAI-1 inhibition exerted by toddalolactone may represent a novel molecular mechanism by which Z. nitidum var. tomentosum manifests its effect in the treatment of thrombosis and fibrosis.

Re: "Plasminogen Activator Inhibitor-1 and Pericardial Fat in Individuals with Type 2 Diabetes Mellitus" by Bayomi et al. (Metab Syndr Relat Disord 2017;15:269-275).

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily, which inactivates tissue plasminogen activator (tPA); therefore, increased level of PAI-1 antigen counteracts the anticoagulant effect of tPA and facilitates the fibrin clot formation. Plasma PAI-1 antigen and activity levels are associated with increased body mass index and with features of the insulin resistance syndrome like obesity and diabetes. Visceral adipose tissue produces more PAI-1 than subcutaneous adipose tissue: This increased production of PAI-1 from the visceral adipose tissue is one important link between visceral obesity and cardiovascular disease. Besides visceral adipose tissue, there is mounting evidence that epicardial adipose tissue may be an important source of PAI-1, especially in patients with type 2 diabetes.

Role of plasminogen activator inhibitor-1 in glucocorticoid-induced muscle change in mice.

We recently revealed that plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is involved in diabetes, osteoporosis and muscle wasting induced by glucocorticoid (GC) treatment in mice. In the present study, we investigated the detailed mechanisms by which GC induces muscle wasting through PAI-1 in vivo and in vitro. PAI-1 deficiency suppressed the mRNA levels of atrogin1 and muscle RING-Finger Protein 1 (MuRF1), ubiquitin ligases leading to muscle degradation, elevated by GC treatment in the gastrocnemius muscle of mice. In vitro study revealed that active PAI-1 treatment augmented the increase in atrogin1 mRNA levels enhanced by dexamethasone (Dex) in mouse myoblastic C2C12 cells. Moreover, a reduction in endogenous PAI-1 level by siRNA suppressed the mRNA levels of atrogin1 and MuRF1 enhanced by Dex in C2C12 cells. In contrast, a reduction in endogenous PAI-1 levels and active PAI-1 did not affect the phosphorylations of Akt and p70S6 kinase nor myogenic differentiation with or without Dex in C2C12 cells. In addition, PAI-1 deficiency blunted IGF-1 mRNA levels decreased by GC treatment in the gastrocnemius muscle of mice, although neither active PAI-1 nor a reduction in endogenous PAI-1 levels affected the levels of IGF-1 mRNA in C2C12 cells in the presence of Dex. In conclusion, our data suggest that paracrine PAI-1 is involved in GC-induced muscle wasting through the enhancement of muscle degradation in mice.

Link between plasminogen activator inhibitor-1 and cardiovascular risk in chronic hepatitis C after viral clearance

The pathophysiological implications of plasminogen activator inhibitor-1 (PAI-1) in HCV infection remain obscure. This prospective study evaluated 669 HCV patients, of whom 536 had completed a course of anti-HCV therapy and had pre-, peri- and post-therapy measurements of various profiles, including PAI-1 levels. Multivariate analysis demonstrated, before anti-HCV-therapy, platelet count and PAI-1-rs1799889 genotype were associated with PAI-1 levels. Among patients with a sustained virological response (SVR, n = 445), platelet count was associated with PAI-1 level at 24 weeks post-therapy. GEE analysis showed that PAI-1-rs-1799889 and interferon-λ3-rs12979860 genotypes affected PAI-1 levels early and late in therapy, respectively. At 24 weeks post-therapy, higher lipid, brain natriuretic peptide, homocysteine and PAI-1 levels and PAI-1 activity were noted only in SVR patients compared with pre-therapy levels. Within 24 weeks post-therapy, 2.2% of the SVR (mean age: 57.8 yr; 8 smoking males; the 2 females had pre-therapy hypercholesteremia or cardiovascular family history of disease) and 0% of the non-SVR patients experienced a new cardiovascular event. Platelet counts consistently correlated with PAI-1 levels regardless of HCV infection. PAI-1-rs-1799889 and interferon-λ3-rs12979860 genotypes mainly affected PAI-1 levels longitudinally. Within 24 weeks post-anti-HCV therapy, the SVR patients showed increasing PAI-1 levels with accelerating cardiovascular risk, especially the vulnerable cases.

Anti-thrombotic and pro-fibrinolytic effects of levosimendan in human endothelial cells in vitro

AimsLevosimendan is an inodilator for the treatment of acute decompensated heart failure (HF). Data from clinical studies suggest that levosimendan is particularly effective in HF due to myocardial infarction. After acute revascularization, no reflow-phenomenon is a common complication that may lead to pump failure and cardiogenic shock. Our aim was to examine whether levosimendan interferes with the pro-thrombotic phenotype of activated endothelial cells in vitro. MethodsHuman heart microvascular endothelial cells (HHMEC) and human umbilical vein endothelial cells (HUVEC) were treated with interleukin-1β (IL-1β) (200U/mL) or thrombin (5U/mL) and co-treated with or without levosimendan (0.1–10μM) for 2–24h. In addition, flow experiments were performed. Effects on plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression and activity were measured by rt-PCR, specific ELISA and flow cytometry. ResultsTreatment with IL-1β or thrombin significantly increased the expression of PAI-1 and TF in endothelial cells. Co-treatment with levosimendan strongly attenuated the effects of IL-1β and thrombin on PAI-1 and TF mRNA by up to 50% and 45%, in a dose- and time-dependent manner. Similar results were obtained under flow conditions. Furthermore, co-treatment with levosimendan dampened the antigen production of PAI-1 and the surface expression of TF by 35% and 45%, respectively. Additionally, levosimendan diminished both TF and PAI-1 activity. ConclusionLevosimendan down-regulates the expression of the pro-thrombotic and anti-fibrinolytic biomolecules TF and PAI-1 in activated human endothelial cells. Our findings may, at least in part, explain some of the beneficial effects of levosimendan after myocardial reperfusion.

The fibrinolytic system: A new target for treatment of depression with psychedelics

Current understanding of the neurobiology of depression has grown over the past few years beyond the traditional monoamine theory of depression to include chronic stress, inflammation and disrupted synaptic plasticity. Tissue plasminogen activator (tPA) is a key factor that not only promotes fibrinolysis via the activation of plasminogen, but also contributes to regulation of synaptic plasticity and neurogenesis through plasmin-mediated activation of a probrain derived neurotrophic factor (BDNF) to mature BDNF. ProBDNF activation could potentially be supressed by competition with fibrin for plasmin and tPA. High affinity binding of plasmin and tPA to fibrin could result in a decrease of proBDNF activation during brain inflammation leading to fibrosis further perpetuating depressed mood. There is a paucity of data explaining the possible role of the fibrinolytic system or aberrant extravascular fibrin deposition in depression. We propose that within the brain, an imbalance between tPA and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) and neuroserpin favors the inhibitors, resulting in changes in neurogenesis, synaptic plasticity, and neuroinflammation that result in depressive behavior. Our hypothesis is that peripheral inflammation mediates neuroinflammation, and that cytokines such as tumor necrosis factor alpha (TNF-α) can inhibit the fibrinolytic system by up- regulating PAI-1 and potentially neuroserpin. We propose that the decrement of the activity of tPA and uPA occurs with downregulation of uPA in part involving the binding and clearance from the surface of neural cells of uPA/PAI-1 complexes by the urokinase receptor uPAR. We infer that current antidepressants and ketamine mitigate depressive symptoms by restoring the balance of the fibrinolytic system with increased activity of tPA and uPA with down-regulated intracerebral expression of their inhibitors. We lastly hypothesize that psychedelic 5-ht2a receptor agonists, such as psilocybin, can improve mood through anti- inflammatory and pro-fibrinolytic effects that include blockade of TNF-α activity leading to decreased PAI-1 activity and increased clearance. The process involves disinhibition of tPA and uPA with subsequent increased cleavage of proBDNF which promotes neurogenesis, decreased neuroinflammation, decreased fibrin deposition, normalized glial-neuronal cross-talk, and optimally functioning neuro-circuits involved in mood. We propose that psilocybin can alleviate deleterious changes in the brain caused by chronic stress leading to restoration of homeostatic brain fibrinolytic capacity leading to euthymia.

Ticagrelor, but not clopidogrel active metabolite, displays antithrombotic properties in the left atrial endocardium.

Oral anticoagulation is considered standard therapy for stroke prevention in atrial fibrillation (AF). Endocardial activation triggers expression of pro-thrombotic mediators including tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), and contributes to thrombus formation in the left atrial appendage (LAA) of AF patients. Recently, pleiotropic effects of specific P2Y12 receptor antagonists were demonstrated; however, whether these drugs possess antithrombotic effects on LAA endocardial cells currently remains unknown. LAA were obtained from 14 patients with known AF undergoing elective cardiac surgery including LAA removal at the University Hospital Zurich. LAA endocardial cells were isolated and pre-incubated with ticagrelor (10-7, 10-6, 10-5M) or clopidogrel active metabolite (CAM) (1.5 × 10-8, 1.5 × 10-7, 1.5 × 10-6 M) before stimulation with tumour necrosis factor-alpha (TNF-α) (10 ng/mL). Finally, TF and PAI-1 expression and activity were analysed. Ticagrelor, unlike CAM, concentration dependently decreased TNF-α-induced TF expression and TF activity in LAA endocardial cells. Further, ticagrelor, but not CAM reduced PAI-1 expression and enzyme activity in TNF-α-stimulated LAA endocardial cells. In contrast, TF pathway inhibitor (TFPI) remained unaffected by both dugs. Ticagrelor, but not CAM, reduces expression and activity of TF and PAI-1 in LAA endocardial cells isolated from patients with AF, indicating possible local antithrombotic effects. Such pleiotropic properties of ticagrelor may contribute to a reduction in thromboembolic complications in patients with AF.

A long-acting PAI-1 inhibitor reduces thrombus formation.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we fused PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which was the H37R variant of PAItrap fused to HSA, gave a six-fold improvement of IC50 (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC50 value of 12 nM, allowing the inhibitor to be evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis.

Reperfusion Shutdown: Delayed Onset of Fibrinolysis Resistance after Resuscitation from Hemorrhagic Shock Is Associated with Increased Circulating Levels of Plasminogen Activator Inhibitor-1 and Postinjury Complications

▪Introduction: In the CRASH II trial, administration of tranexamic acid (TXA) >3 hours after injury was associated with increased mortality. Recent evidence indicates that fibrinolysis inhibition upon presentation to the emergency department (ED) is associated with increased mortality from organ failure. However, it remains unclear if fibrinolysis status changes in response to resuscitation. We hypothesize that the incidence of fibrinolysis resistance (shutdown) increases after resuscitation and is associated with increased post injury complications, prolonged respiratory failure, and prolonged ICU stay.Methods: Blood samples were collected prospectively on injured patients with documented prehospital shock (SBP < 90mm HG ) identified by the emergency medical service as part of an ongoing study to measure the impact of resusciation on coagulation in trauma. Patients had thrombelastography (TEG) samples drawn at admission and sequentially over 24 hours. Susceptibility to fibrinolysis was quantified using a tissue plasminogen activator (tPA) modified TEG. Fibrinolysis shutdown was defined as the lysis at 30 minutes (LY30) < 5thpercentile of healthy volunteers (n=160). Kaplan-Meier plot was generated to identify the timing and cumulative incidence of fibrinolysis shutdown in the first 24 hours after injury. Outcomes were contrasted between patients with and without shutdown at different time intervals from injury using a Mann Whitney U test to assess for 30 day post-injury complications, mechanical ventilator free days, and intensive care unit free days. A nested population of twelve patients that had elevated fibrinolysis activity on presentation to the emergency department and did not receive TXA were analyzed for endogenous tPA and plasminogen activator inhibitor-1(PAI-1) activity during their first 24 hours post injury, which also including pre hospital samples before resuscitation.Results: Ninety patients (median ISS of 27 and prehospital SBP of 72 mmHG) were included in the analysis, with a mortality rate of 13%. Overall, 76 % of patients developed fibrinolysis shutdown. Fifty percent of the population developed fibrinolysis shutdown within 2 hours of injury, and 75% of patients reached shutdown by 4 hours post injury (figure 1). Patients who remained in shutdown at 12 hours postinjury had significantly fewer ventilator free days (median 23 vs 26 p=0.005) and fewer ICU free days (median 20 vs 26 p= 0.004) with longer hospitalization (median 16 days vs 5 p=0.001). Patients in shutdown at 12 hours also had an increased incidence of in hospital complications (40% vs 15% p=0.048). Patients that were in fibrinolysis shutdown at 12 hours had increased plasma (p=0.010) platelet (p=0.007) and TXA (p=0.033) transfusions in the first 6 hours of resuscitation compared to those patients not in shutdown. In the nested cohort of patients with elevated LY30 upon presentation to the hospital, the median tPA activity peaked in prehospital samples (median 0.87 IU/ml) and decreased 10 fold by 4 hours postinjury (0.08 p<0.001), whereas PAI-1 activity increased 700 fold during the same time frame (0.04 to 175 IU/ml p<0.001).Conclusion: Fibrinolysis resistance becomes prevalent in injured patients with prehospital hypotension after successful resuscitation, and this is associated with markedly elevated levels of PAI-1. Patients with fibrinolysis shutdown 12 hours after injury have increased ventilator requirements and prolonged ICU stays. Extending the time patients are in fibrinolysis shutdown during the post resuscitation period appears to be a risk factor for protracted respiratory failure, and may explain why delayed administration of TXA is associated with increased mortality. These results suggest future therapeutic opportunities to reduce PAI-1 activity in the postinjury period to attenuate postinjury organ failure. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development of a plasminogen activator inhibitor (PAI-1) assay and comparison of plasma PAI-1 activity in hyperlipidemic/dyslipidemic dogs with either hyperadrenocorticism or diabetes mellitus, and healthy dogs

Thrombosis is a serious complication of many canine diseases and may be related to decreased fibrinolytic potential. Plasminogen activator inhibitor-1 (PAI-1) is the key regulator of fibrinolysis with increased levels demonstrated in states of pro-thrombosis and abnormal lipid metabolism. Our objective was to develop and validate a canine PAI-1 activity assay and test whether dogs with hyperadrenocorticism or diabetes mellitus that are hyperlipidemic/dyslipidemic have increased plasma PAI-1 activity. Functionally active PAI-1 in the plasma sample was incubated with recombinant tissue plasminogen activator (tPA), allowing the formation of a 1:1 stoichiometric inactive complex. Residual unbound tPA was then reacted with excess plasminogen in the presence of a colorimetric plasmin substrate. Plasmin production is quantified by computing the area under the curve of time (x) vs optical density (y) plot and converted to tPA IU/mL by comparison to a calibration curve of tPA standards. PAI-1 activity was determined by calculating the proportion of exogeneous tPA suppressed by PAI-1 in plasma. Assay verification included assessment of linearity, specificity, precision, sensitivity, and stability. PAI-1 activity was increased in hyperlipidemic compared to healthy dogs, but there was no significant difference between dogs with hyperadrenocorticism and diabetes mellitus. A near significant decrease in activity was detected in thawed plasma stored for 20h at 4°C. Our successfully validated assay offers a new tool for investigating fibrinolysis in dogs. Investigation of PAI-1 activity in dogs with other diseases associated with an increased risk of thrombosis would be valuable. Future studies of PAI-1 activity should consider its lability.

Hyperglycaemia-induced reciprocal changes in miR-30c and PAI-1 expression in platelets.

Type 2 diabetic mellitus (DM2) is associated with accelerated thrombotic complications and is characterized by high levels of plasminogen activator inhibitor-1 (PAI-1). Recent studies show that human platelets have high levels of miR-30c and synthesize considerable active PAI-1. The underlying mechanism of how PAI-1 expression is upregulated in DM2 is poorly understood. We now report that hyperglycaemia-induced repression of miR-30c increases PAI-1 expression and thrombus formation in DM2. Bioinformatic analysis and identification of miRNA targets were assessed using luciferase assays, quantitative real-time PCR and western blots in vitro and in vivo. The changes in miR-30c and PAI-1 levels were identified in platelets from healthy and diabetic individuals. We found that miR-30c directly targeted the 3′ UTR of PAI-1 and negatively regulated its expression. miR-30c was negatively correlated with glucose and HbA1c levels in DM2. In HFD-fed diabetic mice, increasing miR-30c expression by lenti-miR-30c significantly decreased the PAI-1 expression and prolonged the time to occlusion in an arterial thrombosis model. Platelet depletion/reinfusion experiments generating mice with selective ablation of PAI-1 demonstrate a major contribution by platelet-derived PAI-1 in the treatment of lenti-miR-30c to thrombus formation. These results provide important implications regarding the regulation of fibrinolysis by platelet miRNA under diabetic mellitus.

Fasudil inhibits tissue factor and plasminogen activator Inhibitor-1 secretion by peripheral blood mononuclear cells in CAPD patients

Disturbances in hemostasis are common complications of kidney diseases and correlate well with cardiovascular mortality. Little is known about the effects of fasudil on tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) expression in peripheral blood mononuclear cells (PBMCs) in CAPD patients. PBMCs were isolated from 13 individuals with CAPD and 13 healthy subjects. After 4 h of incubation with or without LPS (10 ng/mL), TF and PAI-1 mRNA of PBMCs were detected by RT-PCR. The levels of TF and PAI-1 in culture supernatants of PBMCs were determined by ELISA. Compared with healthy controls, CAPD patients had increased TF, PAI-1 protein and mRNA expression by PBMCs at baseline and after stimulated by LPS (10 ng/mL) [p < 0.001]. The fasudil treatment resulted in a significant effect in decreasing TF and PAI-1 [p < 0.05] synthesis in PBMCs. TF and PAI-1 mRNA expression and activities in PBMCs were increased in CAPD patients. Fasudil reduced LPS-mediated TF and PAI-1 expression and activity in PBMCs. These effects may partially be relevant to the clinical benefits of fasudil in the treatment of CAPD patients.

Modulation of plasminogen activator inhibitor-1 (PAI-1) by the naphthoquinone shikonin

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. Elevated levels of PAI-1 are associated with thrombosis and cardiovascular and metabolic diseases. Inhibition of PAI-1 activity represents a new strategy for antithrombotic and antifibrinolysis therapies. In this study, we systematically investigated the inhibitory effect of shikonin on PAI-1 activity. In the chromogenic substrate-based urokinase (uPA)/PAI-1 assay, we found that shikonin inhibited human PAI-1 activity with IC50 values of 30.68±2.32μM. This result was further confirmed by urokinase-type plasminogen activator (uPA)-mediated clot lysis assay. Mechanistic studies indicated that shikonin directly could bind to PAI-1 and prevent the binding of PAI-1 to uPA in a dose-dependent manner. Shikonin also blocked the formation of PAI-1/uPA complex, as shown by SDS/PAGE analysis. In the mouse arterial thrombosis model, intraperitoneal injection of shikonin at 1mgkg−1 dose significantly prolonged tail bleeding time from 12.956±4.457min to 26.576±2.443min. It also reduced arterial thrombus weight from 0.01±0.001g to 0.006±0.001g (p<0.05). In a liver fibrosis treatment model, when shikonin was continuously injected intraperitoneally at a dose of 1mgkg−1 over a two-week period, the hydroxyproline content in the mice plasma was significantly reduced and the degree of liver fibrosis was decreased significantly. Thus, shikonin may represent a novel small molecule inhibitor of PAI-1 that could have become a lead drug the treatment of thrombus and fibrosis.

Novel Plasminogen Activator Inhibitor-1 Inhibitors Prevent Diabetic Kidney Injury in a Mouse Model

Diabetic nephropathy is the leading cause of end-stage renal disease worldwide, but no effective therapeutic strategy is available. Because plasminogen activator inhibitor-1 (PAI-1) is increasingly recognized as a key factor in extracellular matrix (ECM) accumulation in diabetic nephropathy, this study examined the renoprotective effects of TM5275 and TM5441, two novel orally active PAI-1 inhibitors that do not trigger bleeding episodes, in streptozotocin (STZ)-induced diabetic mice. TM5275 (50 mg/kg) and TM5441 (10 mg/kg) were administered orally for 16 weeks to STZ-induced diabetic and age-matched control mice. Relative to the control mice, the diabetic mice showed significantly increased (p < 0.05) plasma glucose and creatinine levels, urinary albumin excretion, kidney-to-bodyweight ratios, glomerular volume, and fractional mesangial area. Markers of fibrosis and inflammation along with PAI-1 were also upregulated in the kidney of diabetic mice, and treatment with TM5275 and TM5441 effectively inhibited albuminuria, mesangial expansion, ECM accumulation, and macrophage infiltration in diabetic kidneys. Furthermore, in mouse proximal tubular epithelial (mProx24) cells, both TM5275 and TM5441 effectively inhibited PAI-1-induced mRNA expression of fibrosis and inflammation markers and also reversed PAI-1-induced inhibition of plasmin activity, which confirmed the efficacy of the TM compounds as PAI-1 inhibitors. These data suggest that TM compounds could be used to prevent diabetic kidney injury.

Effects of Danhong Injection (丹红注射液) and its main components on anticoagulation and fibrinolysis in cultured vein endothelial cells

To observe the effects of Danhong Injection (丹红注射液) and its main components, including daiclzein and hydroxysafflor yellow A (HSYA), on the anticoagulation, fibrinolysis, anti-apoptosis in hypoxia model of vein endothelial cells (VECs). VECs were prepared and were put in a hypoxia environment, which consisted of mixed gas of 95% N and 5% CO mixed gas, when reached confluent culture. Five groups used different treatments, including normal control group, hypoxia group, daiclzein group, HSYA group and Danhong Injection group. The VECs were identified by fluorescence double labeling methods. The morphology was observed by a phase contrast microscopy. The effects of Danhong Injection, daiclzein and HSYA on 6 keto prostaglandin F1α (6-keto-PGF1α) level was measured by the method of radioimmunoassay (RIA). Superoxide dismutase (SOD) activity was tested by water soluble tetrazolium salt. The content of malondialdehyde (MDA) was measured by thiobarbituric acid. The activities of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were measured by the method of chromogenic substrate. The contents of endothelin (ET) and nitric oxide (NO) were detected by non-equilibrium RIA and enzymelinked immunosorbent assay. Cells apoptosis rate was determined by flow cytometry. Compared with the normal control group, the floating cells number, PAI activity, ET and MDA contents, and cells apoptosis rate in the culture solution of hypoxia group were all significantly increased, whereas the 6-keto-PGF1α and NO contents, and t-PA and SOD activities were decreased significantly (P<0.01). Compared with the hypoxia group, Danhong Injection markedly increased the 6-keto-PGF1α content and SOD activity, regulated PAI and t-PA activities, ET and NO contents, and decreased MDA content and cells apoptosis rate (P<0.05 or P<0.01). Danhong Injection and its main components played an important role in protecting primary VECs from hypoxic damage by regulating the secretion and vasomotor function of VECs. The function of Danhong Injection was most remarkable.

Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts.

Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis.

Infusion of angiotensin II increases fibrinolysis in healthy individuals but not in patients with familial combined hyperlipidemia.

Impaired fibrinolysis is related to insulin resistance, also a characteristic feature of familial combined hyperlipidemia (FCHL). The renin-angiotensin (Ang) system is upregulated with insulin resistance, and there is crosstalk between Ang II and insulin-signalling pathways. We studied the fibrinolytic effects of a 3-h systemic Ang II infusion in 16 patients with FCHL and 16 controls, and placebo infusion in eight individuals. Baseline plasminogen activator inhibitor-1 (PAI-1) activity, plasmin-antiplasmin complex, insulin resistance and C-reactive protein were higher in patients with FCHL than in controls. PAI-1 activity decreased during Ang II, similar in patients with FCHL and controls, and by placebo. Plasmin-antiplasmin complex was unaffected by Ang II in FCHL but increased in controls. Patients with FCHL show signs of insulin resistance, low-grade inflammation and impaired fibrinolysis. Ang II enhances fibrinolysis in controls but not in patients with FCHL, suggesting that patients with FCHL are not capable of increasing tissue plasminogen activator activity in response to Ang II. Ang II has no short-term effects on PAI-1 activity.

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.