Increase In tPA Activity Research Articles

TPA Deficiency Underlies Neurovascular Coupling Dysfunction by Amyloid-β.

The amyloid-β (Aβ) peptide, a key pathogenic factor in Alzheimer's disease, attenuates the increase in cerebral blood flow (CBF) evoked by neural activity (functional hyperemia), a vital homeostatic response in which NMDA receptors (NMDARs) play a role through nitric oxide, and the CBF increase produced by endothelial factors. Tissue plasminogen activator (tPA), which is reduced in Alzheimer's disease and in mouse models of Aβ accumulation, is required for the full expression of the NMDAR-dependent component of functional hyperemia. Therefore, we investigated whether tPA is involved in the neurovascular dysfunction of Aβ. tPA activity was reduced, and the tPA inhibitor plasminogen inhibitor-1 (PAI-1) was increased in male mice expressing the Swedish mutation of the amyloid precursor protein (tg2576). Counteracting the tPA reduction with exogenous tPA or with pharmacological inhibition or genetic deletion of PAI-1 completely reversed the attenuation of the CBF increase evoked by whisker stimulation but did not ameliorate the response to the endothelium-dependent vasodilator acetylcholine. The tPA deficit attenuated functional hyperemia by suppressing NMDAR-dependent nitric oxide production during neural activity. Pharmacological inhibition of PAI-1 increased tPA activity, prevented neurovascular uncoupling, and ameliorated cognition in 11- to 12-month-old tg2576 mice, effects associated with a reduction of cerebral amyloid angiopathy but not amyloid plaques. The data unveil a selective role of the tPA in the suppression of functional hyperemia induced by Aβ and in the mechanisms of cerebral amyloid angiopathy, and support the possibility that modulation of the PAI-1-tPA pathway may be beneficial in diseases associated with amyloid accumulation.SIGNIFICANCE STATEMENT Amyloid-β (Aβ) peptides have profound neurovascular effects that may contribute to cognitive impairment in Alzheimer's disease. We found that Aβ attenuates the increases in blood flow evoked by neural activation through a reduction in tissue plasminogen activator (tPA) caused by upregulation of its endogenous inhibitor plasminogen inhibitor-1 (PAI-1). tPA deficiency prevents NMDA receptors from triggering nitric oxide production, thereby attenuating the flow increase evoked by neural activity. PAI-1 inhibition restores tPA activity, rescues neurovascular coupling, reduces amyloid deposition around blood vessels, and improves cognition in a mouse model of Aβ accumulation. The findings demonstrate a previously unappreciated role of tPA in Aβ-related neurovascular dysfunction and in vascular amyloid deposition. Restoration of tPA activity could be of therapeutic value in diseases associated with amyloid accumulation.

The Markedly Increased PAI1 in Obesity Induces a Compensatory Increase of Hepatocyte Tpa Expression By Activating a LRP1-CREB1 Pathway

Tissue-type plasminogen activator (tPA) initiates fibrinolysis, the primary mechanism that dissolves a thrombus. We have shown that in lean mice the hepatocytes maintain a basal level of circulating tPA that influences fibrinolysis in the occurrence of a vessel injury. As a serine protease, tPA is inhibited by the serpin plasminogen activator inhibitor 1 (PAI1). Both PAI1 and tPA which can be produced by hepatocytes, which is important for regulating energy metabolism and is sensitive to metabolic stress. In obesity, despite an increase in plasma tPA, blood tPA activity and fibrinolysis are reduced primarily due to a larger increase in PAI1. However, the source and regulatory mechanism of this phenomena are unknown. Palmitate treatment of human primary hepatocytes causes an increase in tPA mRNA/protein, reflecting the situation in the obese liver. This increase, however, is overcompensated by a larger increase in PAI1 mRNA, resulting in decreased net tPA activity in the cell culture medium. Silencing PAI1 mRNA with si-SERPINE1 in these cells increased tPA activity in the culture medium. Surprisingly, silencing PAI1 also prevented the increase of tPA mRNA induced by palmitate. Building on this observation, we further treated the cells with active recombinant PAI1 (rPAI1), which induced tPA expression. Next, we fed PAI1fl/flmice with a diet-induced obesity (DIO) diet for three months and silenced their hepatic PAI1 using AAV8-TBG-Cre, which inactivates ~95% floxed gene expression in hepatocytes but not in other cell types of the liver or other tissues. We found that while plasma tPA activity was increased in hepatocyte-PAI1 knockout mice, plasma tPA protein concentration was reduced. In other words, reducing hepatocyte PAI1 in obesity stopped the compensatory increase in liver tPA mRNA and protein. Thus, PAI1 induces tPA, which is likely a compensatory response. To explore the mechanism whereby rPAI1 induces tPA, we considered CREB1, as it is a transcription activator of tPA expression in endothelial cells and is expressed in hepatocytes. We found that CREB1 was activated (phosphorylated) by rPAI1 in human primary hepatocytes and that silencing CREB1 prevented the elevated tPA expression induced by rPAI1. Next, we silenced hepatic CREB1 in obese mice by treating DIO diet-fed CREB1fl/flmice with AAV8-TBG-Cre, which blocked the compensatory increase in tPA and thus worsened the impaired fibrinolysis in obesity. LDL receptor-related protein 1 (LRP1), the major cellular receptor of PAI1, can stimulate CREB1 transcription activity in neurons and adipocytes. As LRP1 is expressed on the surface of hepatocytes, we hypothesized that PAI1 activates CREB1 through LRP1-mediated signaling to increase tPA expression in obesity. Indeed, silencing LRP1 with si-LRP1 stopped the increase of tPA expression induced by rPAI1 in human primary hepatocytes. Interestingly, treating the cells with a mutant rPAI1 lacking the LRP1-interacting heparin-binding domain was unable to induce tPA expression. In summary, hepatocyte tPA mRNA/protein is increased in obesity, but this increase is ultimately overcompensated by a larger increase in PAI1, resulting in decreased plasma tPA activity and fibrinolysis. The markedly increased PAI1, through activating LRP1-CREB1 signaling pathway, drives an increase in hepatic tPA expression as a "compensatory" pathway in obesity. Preventing this compensatory pathway in obesity resulted in the worsening of fibrinolysis. Therapeutic boosting of this compensatory pathway may provide novel strategies to restore effective fibrinolysis in obesity. Disclosures No relevant conflicts of interest to declare.

Efficacy and mechanism of tanshinone IIA liquid nanoparticles in preventing experimental postoperative peritoneal adhesions in vivo and in vitro.

Up to 90% of patients develop adhesion following laparotomy. Upregulating fibrinolysis within the peritoneum reduces adhesions. Tanshinone IIA (Tan IIA) promotes fibrinolysis in hepatic fibrosis and the cardiovascular system and may play a role in preventing adhesions. We report preparation and characterization of liquid nanoparticles of Tan IIA for intravenous administration and investigate its feasibility in clinical practice. Tan IIA liquid nanoparticles (Tan IIA-NPs) were prepared using the emulsion/solvent evaporation method. Adhesions were induced in Sprague–Dawley rats by injuring the parietal peritoneum and cecum, followed by intravenous administration of various Tan IIA-NP dosages. The adhesion scores for each group were collected 7 days after the initial laparotomy. The activity of tissue-type plasminogen activator (tPA) was measured from the peritoneal lavage fluid. The messenger RNA and protein expression levels of plasminogen activator inhibitor-1 (PAI-1) were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. TGF-β1 and collagen I expressions were measured immunohistochemically in the ischemic tissues. The effects of Tan IIA-NPs and free-Tan IIA on tPA and PAI-1 were measured in vitro in TGF-β1-induced HMrSV5 cells. Tan IIA-NPs exhibited small particle size, high encapsulation efficiency, good stability for storage, and safety for intravenous administration. Tan IIA-NPs were effective in preventing adhesion. Tan IIA-NPs increased tPA activity in peritoneal lavage fluid, and tPA mRNA and protein expression, and decreased PAI-1 mRNA and protein expression in the ischemic tissues. Moreover, Tan IIA-NPs decreased TGF-β1 and collagen I expressions in the ischemic tissues. Tan IIA-NPs administered via tail veins upregulated fibrinolysis in the peritoneum. In vitro studies showed that these effects may be mediated by the TGF-β signal pathway.

Resveratrol down-regulates a glutamate-induced tissue plasminogen activator via Erk and AMPK/mTOR pathways in rat primary cortical neurons

Resveratrol (3,5,4'-trihydroxy-trans-stilbene, RSV) is a polyphenolic compound present in a variety of plant species (including grapes) that produces a myriad of biological activities including anti-inflammatory, antioxidant and neuroprotective effects. In this study, we investigate the effects of resveratrol on the basal and glutamate-stimulated expression and activity of a tissue plasminogen activator (tPA) that plays neuromodulatory or neurotoxic roles in many different neurological situations. Under basal conditions, resveratrol decreased the tPA expression and activity without affecting the tPA mRNA level in rat primary cortical neurons. RSV induced AMPK phosphorylation and inhibited mTOR phosphorylation. Inhibition of AMPK phosphorylation using compound C prevented resveratrol-induced down-regulation of tPA activity. This suggested that AMPK/mTOR-dependent translational inhibition contributes to the down-regulation of the tPA. Under glutamate-stimulated conditions of rat primary cortical neurons, tPA activity and expression were increased along with increased tPA mRNA expression but afterward treatment of RSV inhibited the glutamate-induced increase in tPA activity and expression and tPA mRNA expression. Glutamate stimulation induced activation of Akt and MAPK pathways as well as mTOR which were inhibited by RSV. Interestingly, the Erk pathway inhibitor U0126, but neither PI3K-Akt inhibitor LY294002 nor p38 inhibitor SB203580, mimicked the inhibitory action of RSV on glutamate-induced tPA up-regulation. This suggested the essential role of Erk in the transcriptional up-regulation of tPA expression, which is targeted by RSV. Glutamate stimulation induced neuronal cell death as determined by PI staining and MTT assay. However, RSV protected the cultured rat primary cortical neurons from glutamate-induced cell death as paralleled with the changes in tPA expression. These results suggested that RSV can modulate tPA activity under basal and stimulated conditions by both translational and transcriptional mechanisms. The regulation of the tPA by RSV provides additional therapeutic targets on top of the growing number of molecular substrates of RSV's action in the brain.

Diurnal variation of haemostatic response to exercise in young sedentary males.

The aim of this study was to evaluate diurnal variations in the haemostatic response to submaximal exercise performed by young, sedentary men. Fifteen healthy young sedentary males aged 25.6 ± 1.34 (mean ± SD) years performed two exercise sessions, morning and evening, at 70% of maximal oxygen consumption (O2max) on a cycle ergometer for 30 min. Platelet count (PC), activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen, tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) activity were measured as dependent variables. Exercise produced significant increases in PC and fibrinogen for both sessions (P ≤ 0.05), which returned to the resting values after recovery only in the evening session. APTT and PT shortened immediately after exercise, which remained after recovery for both sessions (P ≤ 0.01). Exercise presented significant increases in tPA activity (P ≤ 0.001), which returned to the baseline after recovery in both exercises. PAI-1 activity was significantly higher during the morning than evening (P ≤ 0.05), but no longer demonstrated exercise-related changes. It was found that exercise caused activation of both coagulation and fibrinolysis processes, partly related to the time of the day when the exercise was performed.

Valproic acid induces astrocyte‐dependent neurite outgrowth from cultured rat primary cortical neuron via modulation of tPA/PAI‐1 activity

Tissue plasminogen activator (tPA) is expressed in several regions of brain and plays regulatory roles such as neurite outgrowth, synaptic plasticity and long term potentiation. The activity of tPA is regulated by an endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1), which is expressed mainly in astrocytes. Valproic acid (VPA), a histone deacetylase inhibitor that is used for the treatment of epilepsy and bipolar disorders, promotes neurite extension, neuronal growth and has neuroprotective effect in neurodegenerative diseases. In this study, we examined whether the neurite extension effects of VPA is mediated by modulating tPA/PAI-1 system. VPA dose-dependently increased tPA activity and decreased PAI-1 activity in rat primary astrocytes but not in neurons. PAI-1 protein level secreted into the culture medium but not tPA per se was decreased by VPA. In co-culture system or in neuronal culture stimulated with astrocyte conditioned media but not in pure neuronal cell culture, VPA induced neurite outgrowth via increased tPA activity due to the decreased PAI-1 activity in astrocytes. The decrease in PAI-1 activity and increased neurite extension was regulated via JNK mediated post-transcriptional pathway. The essential role of tPA/PAI-1 system in the regulation of VPA-mediated neurite extension was further demonstrated by experiments using astrocyte conditioned media obtained from tPA or PAI-1 knockout mice. Regulation of PAI-1 activity in astrocyte by VPA may affect both physiological and pathological processes in brain by upregulating tPA activity.

Involvement of tissue plasminogen activator “tPA” in ethanol-induced locomotor sensitization and conditioned-place preference

Ethanol is one of the most abused drugs in the western societies. It is well established that mesolimbic dopaminergic neurons mediate the rewarding properties of ethanol. In our previous studies we have shown that the serine protease tissue plasminogen activator (tPA) is involved in the rewarding properties of morphine and amphetamine. In the current study, we investigated the role of tPA in ethanol-induced behavioral sensitization and conditioned-place preference (CPP). Ethanol treatment dose-dependently induced tPA enzymatic activity in the nucleus accumbens (NAc). In addition, ethanol-induced increase in tPA activity was completely inhibited by pre-treatment with the dopamine D1 and D2 receptor antagonists SCH23390 and raclopride respectively. Furthermore, ethanol-induced locomotor stimulation, behavioral sensitization and conditioned-place preference were enhanced following tPA over-expression in the NAc using a lentiviral vector. In contrast, tPA knock down in the NAc with specific shRNA blocked the rewarding properties of ethanol. The defect of locomotor stimulation in shRNA-injected mice was reversed by microinjections of exogenous recombinant tPA into the nucleus accumbens. Collectively, these results indicate, for the first time, that activation of tPA is effective in enhancing the rewarding effects of ethanol. Targeting the tissue plasminogen activator system would provide new therapeutic approaches to the treatment of alcoholism.

Compartment- and context-specific changes in tissue-type plasminogen activator (tPA) activity following brain injury and pharmacological stimulation

Tissue-type plasminogen activator (tPA) is a major protease of the central nervous system. Most studies to date have used in situ- or gel-based zymographic assays to monitor in vivo changes in neural tPA activity. In this study, we demonstrate that the amidolytic assay can be adapted to accurately detect changes in net tPA activity in mouse brain tissues. Using the amidolytic assay, we examined differences in net tPA activity in the cerebral cortex, sub-cortical structures and cerebellum in wildtype (WT) and tPA−/− mice, and in transgenic mice selectively overexpressing tPA in neurons. In addition, we assessed changes in endogenous net tPA activity in WT mice following morphine administration, epileptic seizures, traumatic brain injury and ischaemic stroke—neurological settings in which tPA has a known functional role. Under these conditions, acute and compartment-specific regulation of tPA activity was observed. tPA also participates in various forms of chronic neurodegeneration. Accordingly, we assessed tPA activity levels in mouse models of Alzheimer's disease (AD) and spinocerebellar ataxia type-1 (SCA1). Decreased tPA activity was detected in the cortex and subcortex of AD mice, whereas increased tPA activity was found in the cerebellum of SCA1 mice. These findings extend the existing hypotheses that low tPA activity promotes AD, whereas increased tPA activity contributes to cerebellar degeneration. Collectively, our results exemplify the utility of the amidolytic assay and emphasise tPA as a complex mediator of brain function and dysfunction. On the basis of this evidence, we propose that alterations in tPA activity levels could be used as a biomarker for perturbations in brain homeostasis.

Biphasic regulation of tissue plasminogen activator activity in ischemic rat brain and in cultured neural cells: Essential role of astrocyte-derived plasminogen activator inhibitor-1

In brain, the serine protease tissue plasminogen activator (tPA) and its endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1) have been implicated in the regulation of various neurophysiological and pathological responses. In this study, we investigated the differential role of neurons and astrocytes in the regulation of tPA/PAI-1 activity in ischemic brain. The activity of tPA peaked transiently and then decreased in cortex and striatum along with delayed induction of PAI-1 in the inflammatory stage after MCAO/reperfusion injury. In cultured primary cells, glutamate stimulation increased tPA activity in neurons but not in other cells such as microglia and astrocytes. With LPS stimulation, a model of neuroinflammatory insults, robust PAI-1 induction was observed in astrocytes but not in neurons and microglia. The upregulation of PAI-1 by LPS in astrocytes was also verified by RT-PCR analysis as well as PAI-1 promoter reporter assay. Lastly, we checked the effects of hypoxia on tPA/PAI-1 activity. Hypoxia increased tPA release from neurons without effects on microglia, while the activity of tPA in astrocyte was decreased consistent with increased PAI-1 activity in astrocyte. Taken together, the results from the present study suggest that neurons are the major source of tPA and that the glutamate-induced stimulated release is mainly governed by neurons in the acute phase. In contrast, the massive up-regulation of PAI-1 in astrocytes during subchronic and chronic inflammatory conditions, leads to decreased tPA activity in the later stages of MCAO. Differential regulation of tPA and PAI-1 in neurons, astrocytes and microglia suggest more attention is required to understand the role of local tPA activity in the vicinity of individual cell types.

Neuroserpin Protects Neurons from Ischemia-Induced Plasmin-Mediated Cell Death Independently of Tissue-Type Plasminogen Activator Inhibition

The serine proteinase tissue-type plasminogen activator (tPA) and the serine proteinase inhibitor neuroserpin are both expressed in areas of the brain with the highest vulnerability to hypoxia/ischemia. In vitro studies show that neuroserpin inhibits tPA and, to a lesser extent, urokinase-type plasminogen activator and plasmin. Experimental middle cerebral artery occlusion (MCAO) increases tPA activity and neuroserpin expression in ischemic tissue, and genetic deficiency of tPA or either treatment with or overexpression of neuroserpin decreases the volume of the ischemic lesion following MCAO. These findings have led to the hypothesis that neuroserpin's neuroprotection is mediated by inhibition of tPA's alleged neurotoxic effect. Ischemic preconditioning is a natural adaptive process whereby exposure to a sublethal insult induces tolerance against a subsequent lethal ischemic injury. Here we demonstrate that exposure to sublethal hypoxia/ischemia increases the neuroserpin expression in the hippocampal CA1 layer and cerebral cortex, and that neuroserpin induces ischemic tolerance and decreases the volume of the ischemic lesion following MCAO in wild-type and tPA-deficient (tPA-/-) neurons and mice. Plasmin induces neuronal death, and this effect is abrogated by either neuroserpin or the NMDA receptor antagonist MK-801. Neuroserpin also attenuated kainic acid-induced neuronal death. Our data indicate that the neuroprotective effect of neuroserpin is due to inhibition of plasmin-mediated excitotoxin-induced cell death and is independent of neuroserpin's ability to inhibit tPA activity.

The effects of whole body vibration and exercise on fibrinolysis in men

Whole body vibration (WBV) is a novel modality that has been demonstrated to enhance muscular and cardiovascular functions reported to increase fibrinolytic activity. The purpose of this study was to examine the fibrinolytic response to WBV and exercise in men. Twenty healthy males (23.8±0.9years, 25.6±0.2kgm(-2)) participated in the study. Each subject performed three trials in randomized order separated by 1week: exercise (X), vibration (V) and vibration+exercise (VX). Exercise sessions consisted of 15min of unloaded squatting at a rate of 20 per minute. Vibration sessions were conducted on a WBV platform vibrating for 15min. Tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were assessed at baseline and immediately after each condition. The increase in tPA activity was significantly greater in VX (0.87±0.35 to 3.21±1.06IUml(-1)) compared to X (0.71±0.36 to 2.4±1.13IUml(-1)) or V (0.83±0.25 to 1.00±0.37IUml(-1)) conditions, and greater in the X condition compared to the V condition. PAI-1 activity decreased significantly more in the VX (6.54±5.53 to 4.89±4.13IUml(-1)) and X (9.76±8.19 to 7.48±7.11IUml(-1)) conditions compared to the V (5.68±3.53 to 5.84±3.52IUml(-1)) condition. WBV does not augment fibrinolytic activity in healthy men. However, WBV combined with squatting exercise increases fibrinolytic activity more than exercise alone.

Memory Self-appraisal and Pet of Brain Amyloid and Tau in Persons Without Dementia

burden, both SDS soluble and insoluble, in the brain of APP/PS1 mice. No significant changes were observed in the levels of full-length APP, alphaor beta-C-terminal fragments; however, TBHQ significantly increased plasma Ab level, suggesting that TBHQ decreases brain Ab burden not by decreasing APP production or altering the processing of APP through aor b-secretases but probably by increasing excretion of Ab from the brain. PAI-1 is a physiological inhibitor of tPA and uPA, which converts plasminogen into plasmin, a serum protease playing an important role in Ab degradation. We showed previously that PAI-1 protein level was increased in the brain of APP/PS1 transgenic mice and in AD patients while knockout of the PAI-1 gene reduced Ab burden in APP/PS1 mice. Here we further showed that TBHQ treatment significantly reduced PAI-1 protein level in the brain of APP/PS1 mice, accompanied by an increase in tPA activity. Conclusions: Together the data suggest that TBHQ treatment reduces brain Ab burden in APP/PS1mice probably by inhibiting PAI-1 gene expression, which leads to increasedAb degradation through plasmin, and by increasing excretion of Ab from the brain. These results suggest a therapeutic value of TBHQ in the treatment of AD.

Fibrinolytic Responses of the Equine Peritoneum to Abdominal Surgery, Surgical Trauma, and Intraperitoneal Sodium Hyaluronate

Postoperative abdominal adhesions are known to present clinical challenges to the surgeon. Adhesion formation is a balance modulated by the fibrinolytic system. The key components involved are the tissue plasminogen activators (tPAs) and plasminogen activator inhibitors (PAI-1 and PAI-2). Sodium hyaluronate (HA) has been shown to reduce the incidence and severity of adhesions in horses. The objectives of this study were to measure tPA and PAI-1 activity in equine peritoneum and evaluate the effect of 0.4% HA solution on local tPA and PAI-1 activity. An exploratory laparotomy was performed and local serosal trauma was induced by using an established abrasion model. Our study involved two groups: in the first group (n = 6) 0.4% HA was used in all intestinal manipulations, whereas in the second group (n = 6) sterile saline was used. Parietal peritoneum, jejunal seromuscular biopsies at abraded sites (AJ) and nonabraded sites, and peritoneal fluid samples were taken at time 0- and at 30-minute intervals up to 120 minutes. Peritoneum tPA activity was significantly decreased at 60 and 90 minutes. Interestingly, AJ contained significantly higher tPA activity than nonabraded sites at 30-, 60-, 90-, and 120-minute intervals in control horses. The increase in tPA activity with AJ in treated (HA) horses was significantly attenuated as compared with the control (saline). Detectable levels of PAI-1 activity could not be identified in our samples. The results of our study indicate that exploratory celiotomy in horses is associated with a significant decrease in peritoneal tPA activity, and HA significantly decreases the fibrinolytic response of the jejunum to surgical trauma. Further characterization of these responses will hopefully lead to new pharmacologic strategies for adhesion prevention.

Tissue-type plasminogen activator is a neuroprotectant in the mouse hippocampus

The best-known function of the serine protease tissue-type plasminogen activator (tPA) is as a thrombolytic enzyme. However, it is also found in structures of the brain that are highly vulnerable to hypoxia-induced cell death, where its association with neuronal survival is poorly understood. Here, we have demonstrated that hippocampal areas of the mouse brain lacking tPA activity are more vulnerable to neuronal death following an ischemic insult. We found that sublethal hypoxia, which elicits tolerance to subsequent lethal hypoxic/ischemic injury in a natural process known as ischemic preconditioning (IPC), induced a rapid release of neuronal tPA. Treatment of hippocampal neurons with tPA induced tolerance against a lethal hypoxic insult applied either immediately following insult (early IPC) or 24 hours later (delayed IPC). tPA-induced early IPC was independent of the proteolytic activity of tPA and required the engagement of a member of the LDL receptor family. In contrast, tPA-induced delayed IPC required the proteolytic activity of tPA and was mediated by plasmin, the NMDA receptor, and PKB phosphorylation. We also found that IPC in vivo increased tPA activity in the cornu ammonis area 1 (CA1) layer and Akt phosphorylation in the hippocampus, as well as ischemic tolerance in wild-type but not tPA- or plasminogen-deficient mice. These data show that tPA can act as an endogenous neuroprotectant in the murine hippocampus.

Effects Of Kaatsu Training On Haemostasis In Healthy Subjects

The KAATSU training is performed under the reduction of muscle blood flow by a specially-designed belt (KAATSU belt), which induces blood pooling in capacitance vessels by restricting venous return. However, no prior studies have examined the effects of KAATSU training on haemostasis. PURPOSE: We investigated acute effects of KAATSU training on haemostasis including fibrinolytic responses under several conditions. in healthy subjects. METHODS: Two protocols have been performed. (1) 6 healthy men (mean age, 48 ± 5 yr) performed KAATSU (160 mmHg) of both thighs for 15 minutes and then KAASTU training combined with low-intensity leg and foot aerobic exercises for ∼10 minutes in hypobaric chamber, which mimics 8000 feet in airflight. (2) Another 7 men (mean age, 30 ± 4 yr) performed leg press exercises (30 % 1 RM) with and without KAATSU of both thighs 24 h after bed rest. Blood samples were taken at rest, immediately after KAATSU, and exercises with or without KAATSU, and after exercise. For the investigation of blood fibrinolysis, determinations of tissue-type plasminogen activator (tPA) activity or antigen, plasminogen activator inhibitor (PAI)-1 activity or antigen, fibrin degradation product (FDP) and D-dimer were used. Prothrombin time (PT) and platelet counts were also measured. RESULTS: (1) In hypobaric chamber, KAATSU by itself significantly increased tPA activity, while PAI-1 activity was unchanged. Furthermore, immediately after the exercise, tPA activity increased significantly. (2) During the exercises combined with KAATSU 24 h after bed rest, tPA antigen significantly increased, compared with control exercises, but PAI-1 antigen was unchanged. In both cases, KAATSU training did not induce fibrin formation as assessed by fibrin D-dimer and FDP. CONCLUSIONS: This study indicates that potentially favorable changes occur in fibrinolytic factors after KAATSU and KAATSU training in healthy subjects.

Differential Regulation of Matrix Metalloproteinase-9 and Tissue Plasminogen Activator Activity by the Cyclic-AMP System in Lipopolysaccharide-stimulated Rat Primary Astrocytes

We investigated the effect of the cAMP system on lipopolysaccharide (LPS)-induced changes in the activity of matrix metalloproteinases (MMPs) and tissue plasminogen activator (tPA) in rat primary astrocytes. LPS stimulation increased MMP-9 and decreased tPA activity in rat primary astrocytes. Co-treatment with a cAMP analog, dibutyryl-cAMP (db-cAMP), or the cAMP elevating beta-adrenergic agonist, isoproterenol, concentration-dependently inhibited LPS-induced MMP-9 activity. In contrast, db-cAMP concentration-dependently increased tPA activity in both basal and LPS-stimulated rat primary astrocytes. To confirm the effect of cAMP on MMP-9 and tPA activity, we treated LPS-stimulated astrocytes with cAMP phosphodiesterase inhibitors, IBMX or rolipram, and they exhibited similar effects to db-cAMP, namely decreasing MMP-9 activity and increasing tPA activity. RT-PCR analysis of MMP-9 mRNA expression and MMP-9 promoter luciferase reporter assays revealed transcriptional upregulation by LPS stimulation and downregulation by db-cAMP. In contrast, the level of tPA mRNA expression was increased both by LPS and by cAMP treatment. Consistent with RT-PCR analysis, tPA promoter reporter assays showed increased activity by both LPS and cAMP stimulation. Interestingly, the level of mRNA encoding plasminogen activator inhibitor-1 (PAI-1) was increased by LPS stimulation and decreased back to control level after co-treatment with db-cAMP, suggesting that PAI-1 expression plays a major role in the regulation of tPA activity. To examine PKA involvement in the effects of db-cAMP on MMP-9 and tPA activity, we added the PKA inhibitors, H89 or rp-cAMP, along with db-cAMP, and they inhibited db-cAMP-mediated changes in tPA activity without affecting MMP-9 activity. These data suggest that cAMP differentially modulates MMP-9 and tPA activity through a mechanism related to PKA activation. The differential regulation of MMP-9 and tPA by the cAMP system may confer more sophisticated regulation of physiological processes, such as extracellular matrix remodeling and cell migration, by activated astrocytes.

Two photon absorption of extended substituted phenylenevinylene oligomers: A TDDFT study

Time dependent density functional theory has been applied to analyze the effect of substituent groups on one-photon (OPA) and two-photon (TPA) absorption properties of extended amine-terminated phenylenevinylene oligomers. All investigated molecules are characterized by increased TPA activity. Calculated TPA cross-sections for these compounds do not show TPA enhancements observed in experimental measurements. Stabilization of the TPA active excited state in these chromophores leads to the small (∼0.2 eV) energy separation between the OPA and TPA allowed excited states, which agrees well with experimental data and may lead to the enhancement of the TPA response due to the strong vibronic couplings.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) in plasminogen activator inhibitor‐1 knockout mice: the effect of fibrinolysis during neuroinflammation

During neuroinflammation in multiple sclerosis (MS) fibrinogen, not normally present in the brain or spinal cord, enters the central nervous system through a compromised blood-brain barrier. Fibrin deposited on axons is ineffectively removed by tissue plasminogen activator (tPA), a key contributory factor being the upregulation of plasminogen activator inhibitor-1 (PAI-1). This study investigated the role of PAI-1 during experimental neuroinflammatory disease. Chronic relapsing experimental allergic encephalomyelitis (CREAE), a model of MS, was induced with spinal cord homogenate in PAI-1 knockout (PAI-1(-/-)) and wild type (WT) mice, backcrossed onto the Biozzi background. Disease incidence and clinical severity were reduced in PAI-1(-/-) mice, with animals developing clinical signs significantly later than WTs. Clinical relapses were absent in PAI-1(-/-) mice and the subsequent reduction in neuroinflammation was coupled with a higher capacity for fibrinolysis in spinal cord samples from PAI-1(-/-) mice, in association with increased tPA activity. Axonal damage was less apparent in PAI-1(-/-) mice than in WTs, implicating fibrin in both inflammatory and degenerative events during CREAE. PAI-1 is a potential target for therapy in neuroinflammatory degenerative diseases, allowing effective fibrin removal and potentially reducing relapse rate and axonal damage.

Fibrinolytic Responses to Resistance Training in Women

During exercise, fibrinolysis increases to protect against exertion-related ischemic events, which are often caused by an occlusive thrombus. No investigation of the fibrinolytic response to resistance training has strictly utilized female participants. This is clinically relevant due to the fact that estrogen, both endogenous and supplemental, has been shown to influence baseline fibrinolytic capacity. Furthermore, fibrinolytic potential is inversely related to body composition. It is unclear if body composition affects the fibrinolytic response to resistance exercise. PURPOSE: The purpose of this study was to assess the fibrinolytic response to resistance training in women, and to assess the effects of body composition on this response. METHODS: Fourteen healthy women (22.6 ± 4 yrs) participated in the study. Subjects were separated into two subsets by body fat as assessed by DEXA: <30% (n=6) and >30% (n=8). Each subject performed 6 sets of 10 leg extension repetitions at an intensity associated with 70% of her measured 1 repetition maximum. The two primary fibrinolytic proteins, tPA and PAI-1, were assessed at baseline, immediately after, and one hour after exercise. Results were analyzed using repeated measures ANOVA. All post-exercise values were corrected for changes in plasma volume. RESULTS: Plasma concentrations of active tPA increased significantly following exercise (p<0.05) and returned to baseline after one hour. Plasma PAI-1 activity did not change following the exercise bout (P<0.05). There was a significant group effect for PAI-1 with the >30% group exhibiting significantly higher plasma concentrations of PAI-1 than the <30% group at all three time points (p<0.05). There were no significant group × time interactions. CONCLUSIONS: Fibrinolytic potential in women increases significantly following a bout of resistance training due to an increase in tPA activity. This cardioprotective response appears to be unrelated to body composition.

Evidence for an Enhanced Fibrinolytic Capacity in Liver Cirrhosis Measured with a New Global Fibrinolysis Test in Whole Blood.

It is known that liver cirrhosis is associated with hyperfibrinolysis, which might contribute to bleeding problems of patients with this disease. However, hyperfibrinolysis has been assessed by using tests (such as euglobulin, plasma or diluted whole blood clot lysis assays), which do not reflect all changes that might occur in the fibrinolytic parameters in blood. A recent study even questioned the presence of a hyperfibrinolytic state in nonbleeding cirrhotic patients, since no evidence of increased fibrinolysis was observed with a new plasma-based clot lysis assay (Gastroenterology 2001; 121:131). Therefore we decided to re-investigate the fibrinolytic state of cirrhotic patients with a recently developed test for global fibrinolytic capacity (GFC) using undiluted whole blood. Non-anticoagulated blood was collected from 30 healthy controls and 75 patients with cirrhosis of varying severity (34 Child-Pugh A, 28 Child-Pugh B and 13 Child-Pugh C). The GFC was determined immediately after collection by clotting the blood, incubating the clots during 3 hours at 37 degrees and then measuring the generated fibrin degradation products. Various haemostatic parameters were determined in plasma samples by using functional activity assays. The median GFC (25th–75th percentile) increased from 1.7 (0.8–3.5) mg/l in the controls to 4.0 (1.6–13.0) mg/l in Child-Pugh A (n.s.), to 11.1 (2.3–31.9) mg/l in Child-Pugh B (P<0.001) and to 22.5 (3.1–89.6) mg/l in Child-Pugh C (P<0.01). Plasma samples of the cirrhotic patients showed significantly decreased levels of antithrombin, plasminogen, alpha-2-antiplasmin and thrombin-activatable fibrinolysis inhibitor (TAFI), significantly increased levels of tissue-type plasminogen activator (tPA) and no significant change in plasminogen activator inhibitor-1 (PAI-1). Spearman correlation tests in the whole group (controls plus patients) showed significant correlations between GFC and antithrombin (r=−0.44), plasminogen (r=−0.38), alpha-2-antiplasmin (r=−0.45), TAFI (r=−0.40), tPA (r=0.77) and PAI-1 (r=−0.51) (all p-values <0.0001). In conclusion, the new GFC test, which includes all components present in blood, indicates that the fibrinolytic capacity is strongly increased in patients with severe cirrhosis. The anti-fibrinolytic effects of decreased plasminogen levels in cirrhosis are apparently overruled by the pro-fibrinolytic effects of decreased alpha-2-antiplasmin and TAFI levels and particularly of increased tPA activity levels.