tPA Activity Research Articles

Plasminogen Activator Inhibitor-1 Mitigates Brain Injury in a Rat Model of Infection-Sensitized Neonatal Hypoxia-Ischemia

Intrauterine infection exacerbates neonatal hypoxic-ischemic (HI) brain injury and impairs the development of cerebral cortex. Here we used low-dose lipopolysaccharide (LPS) pre-exposure followed by unilateral cerebral HI insult in 7-day-old rats to study the pathogenic mechanisms. We found that LPS pre-exposure blocked the HI-induced proteolytic activity of tissue-type plasminogen activator (tPA), but significantly enhanced NF-κB signaling, microglia activation, and the production of pro-inflammatory cytokines in newborn brains. Remarkably, these pathogenic responses were all blocked by intracerebroventricular injection of a stable-mutant form of plasminogen activator protein-1 called CPAI. Similarly, LPS pre-exposure amplified, while CPAI therapy mitigated HI-induced blood-brain-barrier damage and the brain tissue loss with a therapeutic window at 4 h after the LPS/HI insult. The CPAI also blocks microglia activation following a brain injection of LPS, which requires the contribution by tPA, but not the urinary-type plasminogen activator (uPA), as shown by experiments in tPA-null and uPA-null mice. These results implicate the nonproteolytic tPA activity in LPS/HI-induced brain damage and microglia activation. Finally, the CPAI treatment protects near-normal motor and white matter development despite neonatal LPS/HI insult. Together, because CPAI blocks both proteolytic and nonproteolytic tPA neurotoxicity, it is a promising therapeutics of neonatal HI injury either with or without infection.

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

AbstractAlzheimer disease is characterized by the presence of increased levels of the β-amyloid peptide (Aβ) in the brain parenchyma and cerebral blood vessels. This accumulated Aβ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that Aβ42 specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, Aβ42-induced structural changes cannot be the sole mechanism of delayed lysis because Aβ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that Aβ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing Aβ42, and clot lysis by plasmin, but not trypsin, is delayed. Notably, plasmin and tPA activities, as well as tPA-dependent generation of plasmin in solution, are not decreased in the presence of Aβ42. Our results indicate the existence of 2 mechanisms of Aβ42 involvement in delayed fibrinolysis: (1) through the induction of a tighter fibrin network composed of thinner fibers, and (2) through inhibition of plasmin(ogen)–fibrin binding.

TPA Contributes To Aggravation of Endothelin and Thromboxane Induced Cerebrovasoconstriction After Hypoxia/Ischemia Through Upregulation of ERK MAPK

The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, endogenous tPA is upregulated and potentiates impairment of pial artery dilation in response to hypotension after hypoxia/ischemia (H/I) in pigs. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is also upregulated after H/I, with ERK contributing to vasodilator impairment. Impairment of stimulus induced vasodilation may result from tonic withdrawal and/or impairment of a vasodilator influence or upregulation of a vasoconstrictor. This study examined the effect of H/I on the vascular response to two important spasmogens released during CNS ischemic disorders, endothelin‐1 (ET‐1) and thromboxane, and the influence of tPA and ERK MAPK in such responses in pigs equipped with a closed cranial window. Cerebral ischemia was induced via global increase in ICP through saline infusion into a hollow bolt placed in the cranium (dura intact) while hypoxia (pO2 35 mm Hg) was produced by decreasing the inspired O2 via inhalation of N2. H/I aggravated pial artery vasconstriction induced by ET‐1 and the thromboxane mimic U 46619, which was blocked by EEIIMD, an inhibitor of PAI‐1′s vascular activity and signaling of tPA, but not its fibrinolytic action, but unchanged by its inactive analogue EEIIMR. CSF ERK MAPK was increased by H/I and potentiated by tPA. The ERK MAPK antagonist U 0126 blocked H/I induced aggravation of ET‐1 and U 46619 vasoconstriction. These data indicate that H/I aggravates ET‐1 and thromboxane mediated cerebral vasoconstriction through upregulation of tPA and ERK MAPK. These data suggest that thrombolytic therapy for treatment of CNS ischemic disorders can dysregulate cerebrohemodynamics by augmenting vasoconstriction induced by spasmogens co‐released during CNS pathology.

PAI‐1, progress in understanding the clinical problem and its aetiology

Plasminogen activator inhibitor-1 (PAI-1, also known as SERPINE1) is a member of the serine protease inhibitor (SERPIN) superfamily and is the primary physiological regulator of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activity. Although the principal function of PAI-1 is the inhibition of fibrinolysis, PAI-1 possesses pleiotropic functions besides haemostasis. In the quarter century since its discovery, a number of studies have focused on improving our understanding of PAI-1 functions in vivo and in vitro. The use of Serpine1-deficient mice has particularly enhanced our understanding of the functions of PAI-1 in various physiological and pathophysiological conditions. In this review, the results of recent studies on PAI-1 and its role in clinical conditions are discussed.

Abstract 3977: Neuroprotective Effects of Echogenic Liposomes-Mediated Xenon Delivery in a Rat Embolic Stroke Model Combined With Tissue Plasminogen Activator Thrombolysis

BACKGROUND: Ischemic stroke is the second most common cause of death worldwide and a major cause of disability. Intravenous (iv) tissue plasminogen activator (tPA) in combination with the neuroprotective gas, xenon (Xe), is a promising strategy for ischemic stroke treatment. However, Xe delivery by inhalation may reduce oxygen uptake and tPA activity. We have developed novel Xe-containing echogenic liposomes (Xe-ELIP). In this study, we investigated the therapeutic effect of Xe-ELIP in a rat embolic stroke model. METHODS: Xe-ELIP was prepared by the pressurized-freeze method. Thrombotic strokes were induced in male Sprague-Dawley rats (n=16) by injecting a 13mm long blood clot into the middle cerebral artery (MCA). In the treatment group, tPA (10mg/kg) was infused intravenously at 2 hours after the onset occlusion. Xe-ELIP was administrated into the common carotid artery just before iv tPA. Continuous wave ultrasound (1 MHz, 50% duty cycle, 0.5 W/cm 2 ) was applied to trigger Xe release from ELIP during the 5 min of Xe-ELIP administration. Behavioral tests (limb placement, grid walking and beam walking) were conducted three days after the thrombotic stroke. Two mm-thick coronal brain sections were cut and stained with TTC to determine infarct size. RESULTS: The thrombotic stroke control group without any treatment exhibited the largest damage and infarct size (17±5% of the whole brain); tPA treatment reduced the damage and the infarct size to 5.2±0.4% of (p=0.025 vs stroke). tPA treatment in combination with Xe-ELIP further reduced the infarct size to 1.5±0.4% (p=0.05 vs tPA group) ( Fig 1a, 1b). Behavioral deficit correlated with the infarct volume reduction. Regional blood flow velocity monitored by a laser Doppler flow meter was the same in both tPA and tPA+Xe-ELIP treatment groups. CONCLUSIONS: This study has demonstrated a significant neuroprotective effect of ELIP-encapsulated xenon released by application of 1 MHz ultrasound. Xe-ELIP can be used in combination with tPA without affecting tPA thrombolytic activity.

Abstract 2579: tPA Contributes To Aggravation of Endothelin and Thromboxane Induced Cerebrovasoconstriction After Hypoxia/Ischemia Through Upregulation of ERK MAPK

Introduction: The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, endogenous tPA is upregulated and potentiates impairment of pial artery dilation in response to hypotension after hypoxia/ischemia (H/I) in pigs. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is also upregulated after H/I, with ERK contributing to vasodilator impairment. Impairment of stimulus induced vasodilation may result from tonic withdrawal and/or impairment of a vasodilator influence or upregulation of a vasoconstrictor. This study examined the effect of H/I on the vascular response to two important spasmogens released during CNS ischemic disorders, endothelin-1 (ET-1) and thromboxane, and the influence of tPA and ERK MAPK in such responses. Methods: Cerebral ischemia (20 min) was induced via global increase in intracranial pressure via saline infusion into a hollow bolt placed in the cranium (dura intact) while hypoxia (10 min, pO2 35 mm Hg) was produced by decreasing the inspired O2 via inhalation of N2. Vascular responses to topical ET-1 (10-10, 10-8 M) and U 46619 (1, 10 ng/ml) were obtained at 1h post insult. Phosphorylated and total ERK MAPK were measured by ELISA in the CSF of piglets equipped with a closed cranial window. Data (n=5) were analyzed by ANOVA, with significance at p less than 0.05. Results: H/I aggravated pial artery vasconstriction induced by ET-1 and the thromboxane mimic U 46619, which was blocked by EEIIMD, an inhibitor of PAI-1’s vascular activity and signaling of tPA, but not its fibrinolytic action, but unchanged by its inactive analogue EEIIMR. CSF ERK MAPK was increased by H/I and potentiated by tPA. The ERK MAPK antagonist U 0126 blocked H/I induced aggravation of ET-1 and U 46619 vasoconstriction. Discussion: These data indicate that H/I aggravates ET-1 and thromboxane mediated cerebral vasoconstriction through upregulation of tPA and ERK MAPK. These data suggest that thrombolytic therapy for treatment of CNS ischemic disorders can dysregulate cerebrohemodynamics by augmenting vasoconstriction induced by spasmogens co-released during CNS pathology.

Abstract 3475: A Plasminogen Activator Inhibitor-based Therapy of Infection-Sensitized Hypoxia-Ischemia Injury in Newborn Rodent Brains

Introduction: The combination of intrauterine infection and hypoxia-ischemia (HI) is a severe threat to immature brains. Further, infection may alter the response to HI in immature brains, but key steps of the switch are unclear. Better understanding these differential responses may suggest new therapeutic targets in neonatal care. This is particular urgent for premature infants who have a high incidence of chorioamnionitis and HI insults, but are unfit for hypothermia therapy. Hypothesis: (1) Infection pre-exposure significantly amplifies the nuclear factor-kappa B (NF-κB) signaling activity following HI in a tissue-plasminogen activator (tPA)-dependant manner. (2) CPAI, a stable-mutant form of plasminogen activator inhibitor-1 (PAI-1) that resists pure HI, also confers significant protection against infection-sensitized HI brain injury in newborns. Methods: Seven-day-old Wistar rat pups were injected with a low-dose lipopolysaccharide (LPS, 0.3 mg/kg, IP) 4 h before imposing cerebral HI using the Rice-Vannucci model, followed by post-hypoxia injection of CPAI or saline into cerebral ventricles. The brains were collected between 4-24 h recovery for biochemical and histological analyses, at 7 d to measure damage, or at 2 months of age for behavioral testing and diffusion tensor imaging of the white-matter (WM). Results: LPS mitigated HI-induced parenchymal tPA activity, but intensified NF-kB signaling, microglia activation, synthesis of chemoattractive cytokines, damage to blood-brain-barrier, and brain tissue loss, which were all significantly ameliorated by the CPAI treatment. The therapeutic window of CPAI was at least 4 h after the LPS/HI insult. CPAI-treated pups also developed near-normal motor and WM functions at 2 months of age. Conclusions: Infection alters the immature brain response to HI by activating tPA-dependent NF-_B signaling but diminishing the tPA proteolytic activity. Because CPAI effectively inhibits both proteolytic and pro-inflammatory functions of tPA, it may be a promising therapeutic of infection-sensitized neonatal HI brain injury.

Dopamine D3 receptor modulates tissue type plasminogen activator (tPA) activity in mouse brain

Growing amount of evidence points to the dopamine D3 receptor (D3R) as an important mediator in the broad array of events that regulate memory function, perhaps through the modulation of molecular pathways involved in neurotrophic factor activation. Tissue type plasminogen activator (tPA) is a proteolytic enzyme that cleaves the precursor of brain derived neurotrophic factor (proBDNF) into the biologically active form of mature BDNF. However, whether D3Rs modulate tPA activity on BDNF in brain has not been ascertained yet. Here in the present study, using D3R knock-out (D3-/-) mice, we demonstrate that receptor inactivation is associated with increased tPA expression both in prefrontal cortex and, to a greater extent, in the hippocampus, two regions associated with memory processes. The heightened tPA levels observed in D3-/- mice inversely correlated with proBDNF protein expression, whereas they positively correlated with both BDNF mRNA and mature BDNF protein levels. In conclusion, our finding strongly suggest that D3Rs might modulate tPAmediated post-transcriptional processing of BDNF in brain regions critical to memory function.

Therapeutic Value of Small Molecule Inhibitor to Plasminogen Activator Inhibitor–1 for Lung Fibrosis

Fibrosis is a final stage of many lung diseases, with no effective treatment. Plasminogen activator inhibitor-1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA, respectively), plays a critical role in the development of fibrosis. In this study, we explored the therapeutic potential of an orally effective small molecule PAI-1 inhibitor, TM5275, in a model of lung fibrosis induced by transforming growth factor-β1 (TGF-β1), the most potent and ubiquitous profibrogenic cytokine, and in human lung fibroblasts (CCL-210 cells). The results show that an intranasal instillation of AdTGF-β1(223/225), an adenovirus expressing constitutively active TGF-β1, increased the expression of PAI-1 and induced fibrosis in murine lung tissue. On the other hand, treating mice with 40 mg/kg of TM5275 for 10 days, starting 4 days after the instillation of AdTGF-β1(223/225), restored the activities of uPA and tPA and almost completely blocked TGF-β1-induced lung fibrosis, as shown by collagen staining, Western blotting, and the measurement of hydroxyproline. No loss of body weight was evident under these treatment conditions with TM5275. Furthermore, we show that TM5275 induced apoptosis in both myofibroblasts (TGF-β1-treated) and naive (TGF-β1-untreated) human lung fibroblasts, and this apoptosis was associated with the activation of caspase-3/7, the induction of p53, and the inhibition of α-smooth muscle actin, fibronectin, and PAI-1 expression. Such an inhibition of fibrotic responses by TM5275 occurred even in cells pretreated with TGF-β1 for 6 hours. Together, the results suggest that TM5275 is a relatively safe and potent antifibrotic agent, with therapeutic potential in fibrotic lung disease.

Effect of neuroserpin in a neonatal hypoxic-ischemic injury model ex vivo

Hypoxia-ischemia (HI) occurring in immature brains stimulates the expression of tissue-type plasminogen activator (tPA). Neuroserpin is a selected inhibitor of tPA in the central nerves system. However, the role that neuroserpin plays and the possible mechanisms involved during neonatal HI are poorly defined. In this study, an oxygen-glucose deprivation and reoxygenation (OGD/R) model was generated with cultured rat cortical neurons mimicking neonatal HI injury ex vivo, and an acute neuronal excitatory injury was induced by exposure to a high concentration of N-methyl-D-aspartic acid (NMDA). Cells received either neuroserpin or MK-801, an antagonist of the NMDA receptor, during OGD/R, and were incubated with or without neuroserpin after NMDA exposure. Cell viability and morphology were detected by a Cell Counting Kit-8 and immunohistochemical staining, respectively. TPA expression and activity were also assessed. We found that MK-801 alleviated injuries induced by OGD/R, suggesting an excitatory damage involvement. Neuroserpin provided a dose-dependent neuroprotective effect in both OGD/R and acute excitatory injuries by inhibiting the activity of tPA, without affecting neuronal tPA expression. Neuroserpin protected neurons against OGD/R even after a delayed administration of 3h. Collectively, our data indicate that neuroserpin protects neurons against OGD/R. mainly by inhibiting tPA-mediated acute neuronal excitotoxicity.

Effect of Plasminogen Activator Inhibitor-1 and Tissue Plasminogen Activator Polymorphisms on Susceptibility to Type 2 Diabetes in Malaysian Subjects

Elevated activity of plasminogen activator inhibitor-1 (PAI-1) and decreased tissue plasminogen activator (tPA) activity are considered to be important risk factors for type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS). The aim of this study was to investigate the association of the PAI-1 4G/5G and tPA Alu-repeat I/D polymorphisms with T2DM in Malaysian subjects. Serum insulin, coronary risk panel, plasma glucose, and PAI-1 4G/5G and tPA Alu-repeat I/D polymorphisms were studied in 303 T2DM subjects (227 with MetS and 76 without MetS) and 131 normal subjects without diabetes and MetS. Statistical analysis showed that the dominant and additive models of PAI-1 4G/5G polymorphism showed a weak association with T2DM without MetS (OR = 2.35, P = 0.045; OR = 1.67, P = 0.058). On the other hand, the recessive model of the tPA Alu-repeat I/D polymorphism showed an association with T2DM with MetS (OR = 3.32, P = 0.013) whereas the dominant and additive models of the tPA Alu-repeat I/D polymorphism were not associated with T2DM either with or without MetS.

Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 gene expression in cultured mouse Sertoli cells.

We have demonstrated for the first time that (i) mouse Sertoli cells predominantly secrete tPA under the action of FSH and cAMP-generating agents, whereas Leydig cells mainly produce uPA; (ii) Sertoli cells are also capable of secreting PAI-1, as well as FSH, growth factors and GnRH increase PAI-1 gene expression; (iii) the increases in tPA and PAI-1 activities by different hormones in the conditioned media of Sertoli cells correspond to the increases in the levels of tPA and PAI-1 mRNA in the cultured cells, suggesting that the synthesis of the activator-inhibitor in mouse Sertoli cells is regulated at a transcriptional level and the tPA secreted by Sertoli cells may be involved in the spermatogenesis.

Effect of Subconjunctivally Injected Liposome-Encapsulated Tissue Plasminogen Activator on the Absorption Rate of Subconjunctival Hemorrhages in Rabbits

To evaluate the effect of subconjunctivally injected liposome-encapsulated tissue plasminogen activator (tPA) on the absorption rate of subconjunctival hemorrhages (SHs). SHs were induced in 1 eye each of 36 rabbits by subconjunctival injection of 0.05 mL of autologous blood. After 8 hours, randomized subconjunctival injections were performed: 26,000 IU/mL liposome-encapsulated tPA (0.05 mL) in 9 eyes (group A), free-form tPA (26,000 IU/mL; 0.05 mL) in 9 eyes (group B), only liposomes (0.05 mL) in 9 eyes (group C), or no injection in 9 eyes (group D). The sizes of the SHs at 8, 24, 48, 72, 96, and 120 hours after induction were measured using an image analyzer and were compared among the 4 groups. Group A showed significantly more rapid absorption rates than all the other groups at 24, 48, and 72 hours and had the significantly shortest mean elapsed time for the complete resorption of SHs. The tPA activity in ocular tissue except conjunctiva and plasma were negligible beyond 24 hours after SH induction with both forms of tPA. In the conjunctiva, the tPA activity was significantly prolonged in the liposome-encapsulated tPA group than in the free tPA group. Subconjunctival injection of liposome-encapsulated tPA seems to enhance SH absorption in rabbits, especially during the early stages with minimal systemic and ocular absorption.

Microparticles in Acute Promyelocytic Leukemia,

Abstract 3346Abnormal hemostatic function in acute promyelocytic leukemia (APL) contributes to the bleeding and thrombotic complications, which account for much of the morbidity and mortality. Increased procoagulants (tissue factor (TF) and cancer procoagulant) and increased fibrinolytic components (annexin A2 and tPA) have been found in the blood of APL patients and in APL cell line NB4. However, their role in the pathogenesis of these complications is not clear. In recent years, increased numbers of circulating microparticles (MP) containing tissue factor have been observed in many cancer patients. This abstract describes the first study of MP in APL.Plasma samples were collected from 23 APL patients with 37 age-matched controls from healthy subjects. The APL patients were treated with a regimen of ATRA and anthracyclin according to the protocol of the International Consortium for APL (Sem. Thromb. Hemost. 2010, 369:17–924).The first sample was collected at diagnosis and second after induction therapy when molecular remission was obtained in all patients in this study. Platelet poor plasma (PPP) was obtained by centrifuging at 1,500 G for 20 minutes. 250 μL of PPP was centrifuged at 20,000 G for 10 minutes. The sediment containing MP was resuspended in 100μL of trisHCL buffer for fluorochrome-labelling with antibodies respectively against (1) human TF, (2) human annexin A2, (3) CD33, (4) human tPA, (5) human PAI-1 and (6) human annexin A5. Following incubation for 45 min, buffer was added to the suspension to a volume of 0.5 ml for FAC analysis using Coulter CyAn with Summit software. Electronic triggering was done on side-scatter, and samples were gated for size using enumeration beads (0.3 to 1.0 micron) and for annexin A5 positivity. Using MP sediment, the activities of TF, tPA and PAI-1 were respectively measured using chromogenic assays (Actichrome™ TF ELISA, American Diagnostica),(Spectrolyze™ tPA) and (Spectrolyze™ PAI-1).At diagnosis, FAC analysis showed tissue factor, PAI-1 and tPA in the MPs were significantly higher than controls with respective P values of 0.008, 0.0001 and 0.002. MPs with annexin A2 and CD33 in APL samples were not different from controls. After achieving molecular remission, the levels of tissue factor, CD33, and annexin A2 were significantly reduced with respective P values of 0.0001, 0.0007 and 0.05, while those of PAI-1 and tPA were not. Of the MP bearing tissue factor, 83% were associated with MP with CD33. This fraction was high at diagnosis and significantly reduced at remission (P=0.016).On the other hand, when assayed for activity, the TF was functional in all samples, but elevated in only one patient at diagnosis. Likewise, PAI-1 and tPA were functional but their respective activities were not elevated. Western blot showed formation of tPA/PAI-1 complex.These results showed that the increased number of TF-bearing MPs in APL plasma must be viewed with caution. Although the TF was functional, but its activity was not higher than in the controls. The same is true with PAI-1 and tPA. We hypothesize that inhibitors of TF, such as tissue factor pathway inhibitor, are present in APL patients mitigating the hypercoagulability, and that PAI-1 is complexed with tPA, reducing both their activities. Further studies testing this hypothesis are in progress. Disclosures:No relevant conflicts of interest to declare.

Ambient temperature affects thrombotic potential at rest and following exercise

IntroductionDuring exercise, ischemic risk increases, possibly due to changes in coagulation and fibrinolytic activity. Previous research suggests ambient temperature affects resting thrombotic potential, but the effect of heat and cold on hemostasis during exercise is unknown. The purpose of this study was to assess changes in coagulation and fibrinolysis during maximal exercise in hot and cold temperatures, and to compare those responses to exercise under temperate conditions. Materials & MethodsFifteen healthy men completed maximal exercise tests in hot (30°C), temperate (20°C) and cold (5° - 8°C) temperatures. Blood samples were obtained before and immediately after exercise and analyzed for concentrations of thrombin-antithrombin III (TAT), active tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Results were analyzed by ANOVA. ResultsA main effect of time was observed for TAT (temperate=1.71±0.82 – 2.61±0.43ng/ml, hot=1.81±0.73 – 2.62±0.67ng/ml, cold=2.33±0.65 – 2.89±0.81ng/ml, PRE to POST, respectively) and tPA activity (temperate=0.72±0.44 – 2.71±0.55IU/ml, hot=0.72±0.38 – 2.64±0.61IU/ml, cold=0.86±0.45 – 2.65±0.77IU/ml, PRE to POST, respectively). A trend was observed for the PAI-1 response to exercise (temperate=14.5±23.7 – 12.3±20.2IU/ml, hot=15.1±26.5 – 10.0±15.1IU/ml, cold=10.5±10.4 – 7.9±9.7IU/ml, PRE to POST, respectively, p=0.08). TAT concentrations were significantly higher in cold compared to temperate and hot conditions. ConclusionCoagulation potential is elevated during exposure to cold temperatures. These data suggest that risk of an ischemic event may be elevated in the cold.

THR-18, a 18-mer peptide derived from PAI-1, is neuroprotective and improves thrombolysis by tPA in rat stroke models

Objective: The thrombolytic treatment of stroke is limited by a narrow therapeutic time window and is associated with significant adverse side effects. To improve this situation, the modulation of tissue-type plasminogen activator (tPA) activity by a synthetic plasminogen activator inhibitor-1-derived 18-mer peptide (THR-18) was examined in two models of stroke in rats.Methods: In the first model (thromboembolic), stroke was induced by intra-carotid injection of micro-clots to rats, and tPA (6 mg/kg) was intravenously infused for 30 minutes with or without THR-18 (1 mg/kg) at 4 hours post-induction. In the second model [transient middle cerebral artery occlusion (tMCAO)], stroke was induced for 2 hours by a transient mechanical occlusion. tPA and/or THR-18 (0·02, 0·1, and 1 mg/kg) were intravenously infused for 60 minutes at the time of reperfusion.Results: In the thromboembolic model, cerebral blood flow, measured before and up to 5·5 hours post-induction, revealed that tPA administration caused reperfusion of flow at 30 minutes post-infusion. Later on, an additional increase in reperfusion was seen in the tPA+THR-18 group, and not with tPA alone. In both models, the frequency of intracranial hemorrhage in the tPA-treated group was found to be significantly higher than the control, and this tPA effect was attenuated by THR-18. In the thromboembolic study, infarct size and brain edema were similar in the control and tPA-treated rats. However, the combination of tPA and THR-18 caused a statistically significant reduction in both parameters (infarct size 17·8 versus 25·0%, brain edema 5 versus 8%, tPA+THR-18 versus control, respectively). In the tMCAO mechanical model, infarct size and brain edema were both increased by tPA treatment as compared to the control group, and this increase was markedly diminished by THR-18 co-administration. Neurobehavioral assessment of the tMCAO animals performed at 72 hours post-stroke induction revealed significant improvements (P<0·05-0·01) in neuroscores in all groups of animals treated with peptide-tPA, as compared to the tPA monotherapy group. A significant (P<0·05) improvement in the neurological outcome was also seen in the THR-18 monoterapy group, as compared to the control animals, thus demonstrating a clear neuroprotective effect by the peptide on its own.Discussion: The results support the use of THR-18 together with tPA in the thrombolytic therapy of stroke, in order to achieve better patency, less tPA-induced damage, and possibly a widening of tPA therapeutic time window.

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.

Surface-retained tPA is essential for effective fibrinolysis on vascular endothelial cells

AbstractIn a previous study, we demonstrated unique secretory dynamics of tissue plasminogen activator (tPA) in which tPA was retained on the cell surface in a heavy chain–dependent manner after exocytosis from secretory granules in vascular endothelial cells. Here, we examined how retained tPA expresses its enzymatic activity. Retained tPA effectively increased the lysine binding site–dependent binding of plasminogen on the cell surface and pericellular area; this was abolished by inhibition of enzymatic activity of either tPA or plasmin, which suggests that de novo generation of carboxyl-terminal lysine as a consequence of degradation of surface/pericellular proteins by plasmin is essential. Retained tPA initiated zonal clot lysis of a fibrin network that had been formed on vascular endothelial cells, which was preceded by the binding of plasminogen to the lysis front. Our results provide evidence that secreted and retained tPA is essential for maintaining both high fibrinolytic activity and effective clot lysis on the vascular endothelial cell surface.

063 Acceleration of fibrinolysis by factor xa-antithrombin complex

BackgroundThe current model of fibrinolysis accepts fibrin as the only accelerator of tissue plasminogen activator (tPA),-mediated conversion of plasminogen to plasmin, the direct clot-dissolving protease. Our previous work has demonstrated that purified coagulation factor Xa (FXa) undergoes functional modulation by plasmin. In the presence of anionic phospholipid and calcium, under non-reducing conditions, non-covalently associated FXa fragments of 33kDa and 13kDa are generated. Contrary to fibrinolysis dogma, generation of Xa33/13 exposes C-terminal lysine binding sites for plasminogen and tPA, thus converting FXa into a fibrinolysis accelerator. Here we investigated whether Xa33/13 is produced in plasma when FXa has irreversibly associated with its inhibitor, antithrombin (AT), and if the product acquires fibrinolysis accelerator function.Methods/resultsWestern blot analysis of clotting plasma revealed the appearance of Xa33 antigen correlating to the appearance of Xa13-AT. Purified Xa-AT was cleaved approximately 40-fold faster by plasmin compared to FXa. The resulting species has slightly different cleavage sites as determined by N-terminal sequencing, but predicted exposure of new C-terminal lysine. Immobilized Xa33/13-AT bound 125I-plasminogen. To follow purified plasminogen activation by tPA, a chromogenic assay demonstrated Xa-AT enhanced tPA activity several fold over FXa. An excess of Xa-AT in plasma to overcome the endogenous FXa and AT significantly enhanced the rate of fibrinolysis.ConclusionThese data demonstrate a novel function for the Xa-AT complex and support the concept for auxiliary cofactors in fibrinolysis.Heart and Stroke Foundation of British Columbia & Yukon BackgroundThe current model of fibrinolysis accepts fibrin as the only accelerator of tissue plasminogen activator (tPA),-mediated conversion of plasminogen to plasmin, the direct clot-dissolving protease. Our previous work has demonstrated that purified coagulation factor Xa (FXa) undergoes functional modulation by plasmin. In the presence of anionic phospholipid and calcium, under non-reducing conditions, non-covalently associated FXa fragments of 33kDa and 13kDa are generated. Contrary to fibrinolysis dogma, generation of Xa33/13 exposes C-terminal lysine binding sites for plasminogen and tPA, thus converting FXa into a fibrinolysis accelerator. Here we investigated whether Xa33/13 is produced in plasma when FXa has irreversibly associated with its inhibitor, antithrombin (AT), and if the product acquires fibrinolysis accelerator function. The current model of fibrinolysis accepts fibrin as the only accelerator of tissue plasminogen activator (tPA),-mediated conversion of plasminogen to plasmin, the direct clot-dissolving protease. Our previous work has demonstrated that purified coagulation factor Xa (FXa) undergoes functional modulation by plasmin. In the presence of anionic phospholipid and calcium, under non-reducing conditions, non-covalently associated FXa fragments of 33kDa and 13kDa are generated. Contrary to fibrinolysis dogma, generation of Xa33/13 exposes C-terminal lysine binding sites for plasminogen and tPA, thus converting FXa into a fibrinolysis accelerator. Here we investigated whether Xa33/13 is produced in plasma when FXa has irreversibly associated with its inhibitor, antithrombin (AT), and if the product acquires fibrinolysis accelerator function. Methods/resultsWestern blot analysis of clotting plasma revealed the appearance of Xa33 antigen correlating to the appearance of Xa13-AT. Purified Xa-AT was cleaved approximately 40-fold faster by plasmin compared to FXa. The resulting species has slightly different cleavage sites as determined by N-terminal sequencing, but predicted exposure of new C-terminal lysine. Immobilized Xa33/13-AT bound 125I-plasminogen. To follow purified plasminogen activation by tPA, a chromogenic assay demonstrated Xa-AT enhanced tPA activity several fold over FXa. An excess of Xa-AT in plasma to overcome the endogenous FXa and AT significantly enhanced the rate of fibrinolysis. Western blot analysis of clotting plasma revealed the appearance of Xa33 antigen correlating to the appearance of Xa13-AT. Purified Xa-AT was cleaved approximately 40-fold faster by plasmin compared to FXa. The resulting species has slightly different cleavage sites as determined by N-terminal sequencing, but predicted exposure of new C-terminal lysine. Immobilized Xa33/13-AT bound 125I-plasminogen. To follow purified plasminogen activation by tPA, a chromogenic assay demonstrated Xa-AT enhanced tPA activity several fold over FXa. An excess of Xa-AT in plasma to overcome the endogenous FXa and AT significantly enhanced the rate of fibrinolysis. ConclusionThese data demonstrate a novel function for the Xa-AT complex and support the concept for auxiliary cofactors in fibrinolysis.Heart and Stroke Foundation of British Columbia & Yukon These data demonstrate a novel function for the Xa-AT complex and support the concept for auxiliary cofactors in fibrinolysis.

Multipotent Mesenchymal Stromal Cells Increase tPA Expression and Concomitantly Decrease PAI-1 Expression in Astrocytes through the Sonic Hedgehog Signaling Pathway after Stroke (in vitro Study)

Multipotent mesenchymal stromal cells (MSCs) increase tissue plasminogen activator (tPA) activity in astrocytes of the ischemic boundary zone, leading to increased neurite outgrowth in the brain. To probe the mechanisms that underlie MSC-mediated activation of tPA, we investigated the morphogenetic gene, sonic hedgehog (Shh) pathway. In vitro oxygen and glucose deprivation and coculture of astrocytes and MSCs were used to mimic an in vivo ischemic condition. Both real-time-PCR and western blot showed that MSC coculture significantly increased the Shh level and concomitantly increased tPA and decreased plasminogen activator inhibitor 1 (PAI-1) levels in astrocytes. Inhibiting the Shh signaling pathway with cyclopamine blocked the increase of tPA and the decrease of PAI-1 expression in astrocytes subjected to MSC coculture or recombinant mouse Shh (rm-Shh) treatment. Both MSCs and rm-Shh decreased the transforming growth factor-β1 level in astrocytes, and the Shh pathway inhibitor cyclopamine reversed these decreases. Both Shh-small-interfering RNA (siRNA) and Glil-siRNA downregulated Shh and Gli1 (a key mediator of the Shh transduction pathway) expression in cultured astrocytes and concomitantly decreased tPA expression and increased PAI-1 expression in these astrocytes after MSC or rm-Shh treatment. Our data indicate that MSCs increase astrocytic Shh, which subsequently increases tPA expression and decreases PAI-1 expression after ischemia.