Endogenous Tissue Plasminogen Activator Research Articles

Insights Into The Effects of Amino Acid Substitutions on The Stability of Reteplase Structure: A Molecular Dynamics Simulation Study.

Reteplase (recombinant plasminogen activator, r-PA) is a recombinant protein designed to imitate the endogenous tissue plasminogen activator and catalyze the plasmin production. It is known that the application of reteplase is limited by the complex production processes and protein's stability challenges. Computational redesign of proteins has gained momentum in recent years, particularly as a powerful tool for improving protein stability and consequently its production efficiency. Hence, in the current study, we implemented computational approaches to improve r-PA conformational stability, which fairly correlates with protein's resistance to proteolysis. The current study was developed in order to evaluate the effect of amino acid substitutions on the stability of reteplase structure using molecular dynamic simulations and computational predictions. Several web servers designed for mutation analysis were utilized to select appropriate mutations. Additionally, the experimentally reported mutation, R103S, converting wild type r-PA into non-cleavable form, was also employed. Firstly, mutant collection, consisting of 15 structures, was constructed based on the combinations of four designated mutations. Then, 3D structures were generated using MODELLER. Finally, 17 independent 20-ns molecular dynamics (MD) simulations were conducted and different analysis were performed like root-mean-square deviation (RMSD), root-mean-square fluctuations (RMSF), secondary structure analysis, number of hydrogen bonds, principal components analysis (PCA), eigenvector projection, and density analysis. Predicted mutations successfully compensated the more flexible conformation caused by R103S substitution, so, improved conformational stability was analyzed from MD simulations. In particular, R103S/A286I/G322I indicated the best results and remarkably enhanced the protein stability. The conformational stability conferred by these mutations will probably lead to more protection of r-PA in protease-rich environments in various recombinant systems and potentially enhance its production and expression level.

Clinical Relevance of Plasma Endogenous Tissue-Plasminogen Activator and Aortic Valve Sclerosis: Performance as a Diagnostic Biomarker.

Background: Aortic valve sclerosis (AVSc), a common precursor to calcific aortic valve disease, may progress into advanced aortic stenosis with hemodynamic instability. However, plasma biomarkers of such a subclinical condition remain lacking. Since impaired fibrinolysis featuring dysregulated tissue plasminogen activator (t-PA) is involved in several cardiovascular diseases, we investigated whether endogenous t-PA was also associated with AVSc.Methods: Plasma t-PA levels were measured in 295 consecutive patients undergoing standard echocardiography and Doppler flow imaging. Multiple logistic regression analyses were used to assess the association between t-PA and AVSc. Receiver operating characteristic curve analysis was performed for determining the diagnostic value of t-PA for AVSc. The performance of adding t-PA to clinical signatures of AVSc was evaluated. Concentration of t-PA was assessed in human sclerotic and non-sclerotic aortic valves by histology and immunohistochemistry analysis.Results: Plasma t-PA was higher in patients with AVSc than in non-AVSc counterparts (median, 2063.10 vs. 1403.17 pg/mL, p < 0.01). C-statistics of plasma t-PA for discriminating AVSc was 0.698 (95%CI: 0.639–0.758). The performance of t-PA for identifying AVSc was better among male and non-hypertensive patients [C-statistics (95%CI): 0.712 (0.634–0.790) and 0.805 (0.693–0.916), respectively]. Combination of t-PA and clinical factors improved classification of the patients (category-free NRI: 0.452, p < 0.001; IDI: 0.020, p = 0.012). The concentration of t-PA was three times higher in sclerotic compared to non-sclerotic aortic valves.Conclusion: Elevated circulating t-PA level confers an increased risk for AVSc. Further prospective studies with larger sample size are needed to examine if t-PA could serve as a diagnostic clinical marker for AVSc.

D1 receptor‐mediated endogenous tPA upregulation contributes to blood‐brain barrier injury after acute ischaemic stroke

Blood‐brain barrier (BBB) integrity injury within the thrombolytic time window is becoming a critical target to reduce haemorrhage transformation (HT). We have previously reported that BBB damage was initially damaged in non‐infarcted striatum after acute ischaemia stroke. However, the underlying mechanism is not clear. Since acute ischaemic stroke could induce a significant increase of dopamine release in striatum, in current study, our aim is to investigate the role of dopamine receptor signal pathway in BBB integrity injury after acute ischaemia using rat middle cerebral artery occlusion model. Our data showed that 2‐h ischaemia induced a significant increase of endogenous tissue plasminogen activator (tPA) in BBB injury area and intra‐striatum infusion of tPA inhibitor neuroserpin, significantly alleviated 2‐h ischaemia‐induced BBB injury. In addition, intra‐striatum infusion of D1 receptor antagonist SCH23390 significantly decreased ischaemia‐induced upregulation of endogenous tPA, accompanied by decrease of BBB injury and occludin degradation. More important, inhibition of hypoxia‐inducible factor‐1 alpha with inhibitor YC‐1 significantly decreased 2‐h ischaemia‐induced endogenous tPA upregulation and BBB injury. Taken together, our data demonstrate that acute ischaemia disrupted BBB through activation of endogenous tPA via HIF‐1α upregulation, thus representing a new therapeutic target for protecting BBB after acute ischaemic stroke.

Inhibiting endogenous tissue plasminogen activator enhanced neuronal apoptosis and axonal injury after traumatic brain injury.

Tissue plasminogen activator is usually used for the treatment of acute ischemic stroke, but the role of endogenous tissue plasminogen activator in traumatic brain injury has been rarely reported. A rat model of traumatic brain injury was established by weight-drop method. The tissue plasminogen activator inhibitor neuroserpin (5 μL, 0.25 mg/mL) was injected into the lateral ventricle. Neurological function was assessed by neurological severity score. Neuronal and axonal injuries were assessed by hematoxylin-eosin staining and Bielschowsky silver staining. Protein level of endogenous tissue plasminogen activator was analyzed by western blot assay. Apoptotic marker cleaved caspase-3, neuronal marker neurofilament light chain, astrocyte marker glial fibrillary acidic protein and microglial marker Iba-1 were analyzed by immunohistochemical staining. Apoptotic cell types were detected by immunofluorescence double labeling. Apoptotic cells in the damaged cortex were detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling staining. Degenerating neurons in the damaged cortex were detected by Fluoro-Jade B staining. Expression of tissue plasminogen activator was increased at 6 hours, and peaked at 3 days after traumatic brain injury. Neuronal apoptosis and axonal injury were detected after traumatic brain injury. Moreover, neuroserpin enhanced neuronal apoptosis, neuronal injury and axonal injury, and activated microglia and astrocytes. Neuroserpin further deteriorated neurobehavioral function in rats with traumatic brain injury. Our findings confirm that inhibition of endogenous tissue plasminogen activator aggravates neuronal apoptosis and axonal injury after traumatic brain injury, and activates microglia and astrocytes. This study was approved by the Biomedical Ethics Committee of Animal Experiments of Shaanxi Province of China in June 2015.

In silico Analysis of Several Signal Peptides for the Excretory Production of Reteplase in Escherichia coli

Background: Solubility of recombinant therapeutic enzymes is one of the major purpose their production in bacterial host The factors such as metabolic system, a cellular stress response of the bacterial host and formation of inclusion bodies cause undesirable expression results. One method to overcome this problem is secretion of recombinant protein into the extracellular growth medium by selection of proper signal peptides. Reteplase (Retavase) is a serine protease simulating endogenous tissue plasminogen activator (t-PA), fibrin-specific plasminogen activator, playing role in thrombolysis and fibrinolysis (clot lysis) by converting plasminogen to plasmin. Objective: The aim of this study was to evaluate several signal peptides in order to selection the proper one which suitable for excretory production of the reteplase. Method: The presence and cleavage site location in signal peptides was predicted by SignalP 4.1. The prediction of secretory pathway of signal peptides was performed using high accurate database. Several critical features of the signal peptide were evaluated applying various online web-servers such as ProtParam and Solpro with high sensitivity and specificity. Finally, the selected signal peptides were compared and the one which was appropriate candidate for high yields production of secretory reteplase in the E. coli host was defined. Results: Among total 30 signal peptides, 6 of them, CHIS_BACSU, MPT53_MYCTU, PET_ECOLX, PIC_ECOL6, PIC_ECLOX, TSH_ECOLX were omitted. CPLR_DESHA, AGAR_ALTAT, CDGT_BACS2, PAG_BACAN could be considered as proper candidate for efficient extracellular production of reteplase in E. coli. Conclusion: Theoretical analysis and evaluation of signal peptides by using computational tools can be acceptably carried out, but these results must be established via practical effort. Keywords: E. coli host, bioinformatics, secretion, physicochemical properties, solubility, signal peptides.

Hemorrhagic Transformation after Ischemic Stroke in Animals and Humans

Hemorrhagic transformation (HT) is a common complication of ischemic stroke that is exacerbated by thrombolytic therapy. Methods to better prevent, predict, and treat HT are needed. In this review, we summarize studies of HT in both animals and humans. We propose that early HT (<18 to 24 hours after stroke onset) relates to leukocyte-derived matrix metalloproteinase-9 (MMP-9) and brain-derived MMP-2 that damage the neurovascular unit and promote blood-brain barrier (BBB) disruption. This contrasts to delayed HT (>18 to 24 hours after stroke) that relates to ischemia activation of brain proteases (MMP-2, MMP-3, MMP-9, and endogenous tissue plasminogen activator), neuroinflammation, and factors that promote vascular remodeling (vascular endothelial growth factor and high-moblity-group-box-1). Processes that mediate BBB repair and reduce HT risk are discussed, including transforming growth factor beta signaling in monocytes, Src kinase signaling, MMP inhibitors, and inhibitors of reactive oxygen species. Finally, clinical features associated with HT in patients with stroke are reviewed, including approaches to predict HT by clinical factors, brain imaging, and blood biomarkers. Though remarkable advances in our understanding of HT have been made, additional efforts are needed to translate these discoveries to the clinic and reduce the impact of HT on patients with ischemic stroke.

Abstract 9533: Retention of Endothelial Progenitor Cells in Bone Marrow in a Murine Model of Endogenous Tissue Plasminogen Activator (tPA) Deficiency in Response to Critical Limb Ischemia

Background: This study tested the hypothesis that tissue plasminogen activator (tPA) is crucial for regulating endothelial progenitor cell (EPC) mobilization from bone marrow to circulation in murine critical limb ischemia (CLI) by ligating left femoral artery. Methods and Results: Wild-type (C57BL/6) (n=40) mice were equally divided into group 1A (sham control), group 2A (CLI), group 3A [control-tPA (4.0 mg/kg, intravenously at 3h after CLI)], group 4A (CLI-tPA). Similarly, tPA knock-out (tPA -/- ) mice (n=40) were equally divided into group 1B (sham control) group 2B (CLI), group 3B [control-tPA (4.0 mg/kg)], group 4B (CLI-tPA). The circulating levels of EPCs (C-kit/CD31+, Sca-1/KDR+, CXCR4/CD34+) were lower in groups 1B and 2B than in groups 1A and 2A, respectively (all p&lt;0.01), and were reversed after tPA treatment (3B vs. 3A or 4B vs. 4A, p&gt;0.05) at 6h and 18h post-CLI. Levels of these biomarkers decreased again 14 days after CLI in tPA -/- mice compared to those in wild-type between the respective groups (all p&lt;0.01). Laser Doppler flowmetry showed a higher ratio of ischemic-to-normal blood flow in 2A than in 2B and in 4A than in 4B by day 14 after CLI (all p&lt;0.05). Angiogenesis at protein (CXCR4, SDF-1α, VEGF) and cellular (CXCR4+, SDF-1α +, and CD31+ cells) levels were highest in animals with CLI-tPA, significantly higher in mice with CLI only than in sham controls for both wild-type and tPA -/- mice (p&lt;0.01). Conclusion: tPA played an essential role in augmenting circulating EPCs, angiogenesis, and blood flow in the ischemic limb in a murine model. Key words: tissue plasminogen activator, critical limb ischemia, angiogenesis, circulating endothelial progenitor cells

Retention of endothelial progenitor cells in bone marrow in a murine model of endogenous tissue plasminogen activator (tPA) deficiency in response to critical limb ischemia

BackgroundThis study tested the hypothesis that tissue plasminogen activator (tPA) is crucial for regulating endothelial progenitor cell (EPC) mobilization from bone marrow to circulation in murine critical limb ischemia (CLI) by ligating the left femoral artery. MethodsWild-type (C57BL/6) (n=40) mice were equally divided into group 1A (sham control), group 2A (CLI), group 3A [control-tPA (4.0mg/kg)] and group 4A [CLI-tPA (intravenously at 3h after CLI)]. Similarly, tPA knock-out (tPA−/−) mice (n=40) were equally divided into group 1B (sham control), group 2B (CLI), group 3B [control-tPA (4.0mg/kg)], and group 4B (CLI-tPA). ResultsThe circulating levels of EPCs (C-kit/CD31+, Sca-1/KDR+, CXCR4/CD34+) were lower in groups 1B and 2B than in groups 1A and 2A, respectively (all p<0.01), and were reversed after tPA treatment (3B vs. 3A or 4B vs. 4A, p>0.05) at 6h and 18h post-CLI. Levels of these biomarkers decreased again 14days after CLI in tPA−/− mice compared to those in wild-type between the respective groups (all p<0.01). Laser Doppler flowmetry showed a higher ratio of ischemic-to-normal blood flow in 2A than in 2B and in 4A than in 4B by day 14 after CLI (all p<0.05). Angiogenesis at protein (CXCR4, SDF-1α, VEGF) and cellular (CXCR4+, SDF-1α+, and CD31+ cells) levels was highest in animals with CLI-tPA, significantly higher in mice with CLI only than in sham controls for both wild-type and tPA−/− mice (p<0.01). ConclusiontPA played an essential role in augmenting circulating EPCs, angiogenesis, and blood flow in the ischemic limb in a murine model.

Tissue plasminogen activator arrests Alzheimer's disease pathogenesis

The progressive deposition of amyloid-β (Aβ) in the brain is a pathologic feature of Alzheimer's disease (AD). This study was aimed to determine whether endogenous tissue plasminogen activator (tPA) modulates the pathogenic process of AD. tPA expression and activity developed around amyloid plaques in the brains of human amyloid precursor protein–overexpressing Tg2576 mice, which were weakened by the genetic ablation of tPA. Although the complete loss of tPA was developmentally fatal to Tg2576 mice, tPA-heterozygous Tg2576 mice expressed the more severe degenerative phenotypes than tPA wild-type Tg2576 mice, including abnormal and unhealthy growth, shorter life spans, significantly enhanced Aβ levels, and the deposition of more and larger amyloid plaques in the brain. In addition, the expression of synaptic function–associated proteins was significantly reduced, which in turn caused a more severe impairment in learning and memory performance in Tg2576 mice. Thus, endogenous tPA, preferentially its aggregate form, could degrade Aβ molecules and maintain low levels of brain Aβ, resulting in the delay of AD pathogenesis.

Anaphylactic shock associated with intravenous thrombolytics

Adverse events including intracerebral hemorrhage and reperfusion arrhythmias are well known to occur with thrombolytic therapy. We report a case report of anaphylactic reaction directly attributable to intravenous (IV) recombinant tissue plasminogen activator and identify additional cases through review of the Food and Drug Administration Adverse Event Reporting System. A systematic review of Adverse Event Reporting System was performed for allergic adverse events occurring in conjunction with IV thrombolytics. We reviewed 924 adverse events which occurred between 2004 and 2012 that were associated with thrombolytics. We subsequently acquired detailed individual safety reports of 33 cases in which allergic events were documented. Out of the 33 reports, there were 12 cases (age range, 57-93 years) of adverse allergic reaction directly attributable to IV thrombolytics. Allergic reactions included angioedema, facial swelling, urticaria, skin rash, cutaneous hypesthesia, hypotension, anaphylactic shock, and death. Of the patients who were reported to suffer from allergic adverse events, 11 received IV alteplase and 1 received IV reteplase. Most reactions associated with IV alteplase resolved with withdrawal of medication and treatment with diphenhydramine and steroids ± epinephrine. There was 1 death directly attributable to allergic reaction in a patient who received IV reteplase for MI.Although IV alteplase is identical to endogenous tissue plasminogen activator, it appears to be the most common cause of allergic reaction among currently used thrombolytics, with or without concomitant administration of angiotensin-converting enzyme inhibitors. A greater awareness among physicians may result in prompt recognition and treatment.

Endogenous tissue plasminogen activator enhances fibrinolysis and limits thrombus formation in a clinical model of thrombosis

Purpose: Fibrinolysis inhibits thrombus propagation on the surface of unstable atherosclerotic plaques. Using a clinical model of deep arterial injury, we assessed the ability of exogenous and endogenous tissue Plasminogen Activator (t-PA) to limit acute in situ thrombus formation. Methods and results: Ex vivo thrombus formation was assessed in the Badimon Chamber at low and high shear rates in two double-blind randomised cross-over studies of 20 healthy volunteers during extracorporeal administration of recombinant t-PA (0, 40, 200 and 1000 ng/mL) or during endogenous t-PA release stimulated by intra-arterial bradykinin infusion in the presence or absence of oral enalapril. Recombinant t-PA caused a dose-dependent reduction in thrombus area under low and high shear conditions (p<0.001 for all). Intra-arterial bradykinin increased plasma t-PA concentrations in the chamber effluent (p<0.01 for all versus saline) that was quadrupled in the presence of enalapril (p<0.0001 versus placebo) (Figure 1, panel A). These increases were accompanied by an increase in plasma D-dimer concentration (p<0.005 for all versus saline) (Figure 1, panel B) and, in the presence of enalapril, a reduction in thrombus area in the low shear (16%±5%, p=0.03) (Figure 1, panel C) and a trend towards a reduction in the high shear chamber (13%±7%, p=0.07) (Figure 1, panel D). ![Figure][1] Figure 1 Conclusions: Using a well-characterised clinical model of coronary arterial injury, we demonstrate that endogenous t-PA released from the vascular endothelium enhances fibrinolysis and limits in situ thrombus propagation. These data support a crucial role for the endogenous fibrinolytic system in vivo and suggest that continued exploration and manipulation of its therapeutic potential is warranted. [1]: pending:yes

Mild Hypothermia Reduces Tissue Plasminogen Activator-Related Hemorrhage and Blood Brain Barrier Disruption After Experimental Stroke

Therapeutic hypothermia has shown neuroprotective promise, but whether it can be used to improve outcome in stroke has yet to be determined in patients. Recombinant tissue plasminogen activator (rt-PA) is only given to a minority of patients with acute ischemic stroke, and is not without risk, namely significant brain hemorrhage.We explored whether mild hypothermia, in combination with rt-PA, influences the safety of rt-PA. Mice were subjected to middle cerebral artery occlusion (MCAO) using a filament model, followed by 24 hours reperfusion.Two paradigms were studied. In the first paradigm, cooling and rt-PA treatment began at the same time upon reperfusion, whereas in the second paradigm, cooling began soon after ischemia onset, and rt-PA began after rewarming and upon reperfusion. Experimental groups included: tPA treatment at normothermia (37°C), rt-PA treatment at hypothermia (33°C), no rt-PA at normothermia, and no rt-PA treatment at hypothermia. Infarct size, neurological deficit scores, blood brain barrier (BBB) permeability, brain hemorrhage, and expression of endogenous tissue plasminogen activator (tPA) and its inhibitor, plasminogen activator inhibitor (PAI-1) were assessed. For both paradigms, hypothermia reduced infarct size and neurological deficits compared to normothermia, regardless of whether rt-PA was given. rt-PA treatment increased brain hemorrhage and BBB disruption compared to normothermia, and this was prevented by cooling. However, mortality was higher when rt-PA and cooling were administered at the same time, beginning 1–2 hours post MCAO. Endogenous tPA expression was reduced in hypothermic mice, whereas PAI-1 levels were unchanged by cooling. In the setting of rt-PA treatment, hypothermia reduces brain hemorrhage, and BBB disruption, suggesting that combination therapy with mild hypothermia and rt-PA appears safe.

Endogenous Tissue Plasminogen Activator Enhances Fibrinolysis and Limits Thrombus Formation in a Clinical Model of Thrombosis

Using a clinical model of deep arterial injury, we assessed the ability of exogenous and endogenous tissue plasminogen activator (t-PA) to limit acute in situ thrombus formation. Ex vivo thrombus formation was assessed in the Badimon chamber at low and high shear rates in 2 double-blind randomized cross-over studies of 20 healthy volunteers during extracorporeal administration of recombinant t-PA (0, 40, 200, and 1000 ng/mL) or during endogenous t-PA release stimulated by intra-arterial bradykinin infusion in the presence or absence of oral enalapril. Recombinant t-PA caused a dose-dependent reduction in thrombus area under low and high shear conditions (P<0.001 for all). Intra-arterial bradykinin increased plasma t-PA concentrations in the chamber effluent (P<0.01 for all versus saline) that was quadrupled in the presence of enalapril (P<0.0001 versus placebo). These increases were accompanied by an increase in plasma D-dimer concentration (P<0.005 for all versus saline) and, in the presence of enalapril, a reduction in thrombus area in the low shear (16±5; P=0.03) and a trend toward a reduction in the high shear chamber (13±7%; P=0.07). Using a well-characterized clinical model of coronary arterial injury, we demonstrate that endogenous t-PA released from the vascular endothelium enhances fibrinolysis and limits in situ thrombus propagation. These data support a crucial role for the endogenous fibrinolytic system in vivo and suggest that continued exploration and manipulation of its therapeutic potential are warranted.

Abstract WP73: Potentially Fatal Adverse Allergic Events to Intravenous Thrombolytics: An analysis from Adverse Event Reporting System

Objective: Adverse events including intracerebral hemorrhage (ICH) and reperfusion arrythmias are known to occur with thrombolytic therapy. Due to limited data, our goal is to describe the types of allergic adverse events directly attributable to IV thrombolytics. Methods: Adverse Event Reporting System (AERS) is a database that contains information on adverse event and medication error reports submitted to FDA. A systematic review of AERS database was performed for allergic adverse events occurring in conjunction with IV thrombolytics, including alteplase, tenecteplase, urokinase and reteplase. Allergic reaction was defined as any non-hemorrhagic sensitivity reaction which occurred as a direct result of administration of IV thrombolytic. We reviewed 924 adverse events which occurred between 2004-2010 associated with thrombolytics and further requested 33 detailed ISR reports of allergic reactions. Results: Out of the 33 reports, there were 12 cases (age range 57 to 93 years) of adverse allergic reaction directly attributable to IV thrombolytics. Allergic reactions included angioedema, facial swelling, urticaria, skin rash, cutaneous hypesthesia, hypotension, seizure, anaphylactic shock and death. Of the patients who were reported to suffer from allergic adverse events 11 received IV alteplase, and 1 received IV reteplase. Four of these patients were taking ACE inhibitors at the time of allergic reaction. In the remaining 21 events, the IV thrombolytics remained as a possible secondary cause of allergic reaction, but concomitantly administered medications made this difficult to ascertain. Most reactions associated with IV alteplase resolved with withdrawal of medication and treatment with diphenhydramine and steroids +/- epinephrine. There was 1 death directly attributable to allergic reaction in a patient who received IV reteplase for MI. Conclusion: Although IV alteplase is identical to endogenous tissue plasminogen activator, it appears to be the most common cause of allergic reaction amongst currently used thrombolytics, with or without concomitant administration of ACE inhibitors. A greater awareness among physicians may result in prompt recognition and treatment.

Immunotherapy blocking the tissue plasminogen activator-dependent activation of N-methyl-d-aspartate glutamate receptors improves hemorrhagic stroke outcome

Ischemic and hemorrhagic strokes have different etiologies, but share some pathogenic mechanisms, including a pro-neurotoxic effect of endogenous tissue plasminogen activator (tPA) via N-methyl-d-Aspartate (NMDA) receptors. Thus, in a model of intracerebral hemorrhage in rats, we investigated the therapeutic value of a strategy of immunotherapy (αATD-GluN1 antibody) preventing the interaction of tPA with NMDA receptors. We found that a single intravenous injection of αATD-GluN1 reduced brain edema, neuronal death, microglial activation and functional deficits following intracerebral hemorrhage, without affecting the hematoma volume.

Combination therapy with glucagon and a novel plasminogen activator inhibitor-1-derived peptide enhances protection against impaired cerebrovasodilation during hypotension after traumatic brain injury through inhibition of ERK and JNK MAPK

Objective: Outcome of traumatic brain injury (TBI) is impaired by hypotension and glutamate, and TBI-associated release of endogenous tissue plasminogen activator (tPA) impairs cerebral autoregulation. Glucagon decreases central nervous system glutamate, lessens neuronal cell injury, and improves neurological score in mice after TBI. Glucagon partially protects against impaired cerebrovasodilation during hypotension after TBI in piglets by upregulating cAMP which decreases release of tPA. Pial artery dilation during hypotension is due to release of cAMP-dependent dilator prostaglandins (PG), such as PGE2 and PGI2. TBI impairs PGE2 and PGI2-mediated pial artery dilation, which contributes to disturbed cerebral autoregulation post-insult, by upregulating mitogen-activated protein kinase (MAPK). This study was designed to investigate relationships between tPA, prostaglandins, and MAPK as a mechanism to improve the efficacy of glucagon-mediated preservation of cerebrovasodilation during hypotension after TBI.Methods: Lateral fluid percussion brain injury (FPI) was induced in piglets equipped with a closed cranial window. ERK and JNK MAPK concentrations in cerebrospinal fluid were quantified by enzyme-linked immunosorbent assay.Results: Cerebrospinal fluid JNK MAPK was increased by FPI, but blunted by glucagon and the novel plasminogen activator inhibitor-1-derived peptide (PAI-1DP), Ac-RMAPEEIIMDRPFLYVVR-amide. FPI modestly increased, while glucagon and PAI-1DP decreased ERK MAPK. PGE2, PGI2, N-methyl-D-aspartate, and hypotension-induced pial artery dilation was blunted after FPI, partially protected by glucagon, and fully protected by glucagon+PAI-1DP, glucagon+JNK antagonist SP600125 or glucagon+ERK inhibitor U 0126.Discussion: Glucagon+PAI-1DP act in concert to protect against impairment of cerebrovasodilation during hypotension after TBI via inhibition of ERK and JNK MAPK.

Platelet Protease Nexin-1, a Serpin That Strongly Influences Fibrinolysis and Thrombolysis

Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma, but we have shown recently that PN-1 is present within the α-granules of platelets. In this study, the role of platelet PN-1 in fibrinolysis was investigated with the use of human platelets incubated with a blocking antibody and platelets from PN-1-deficient mice. We showed by using fibrin-agar zymography and fibrin matrix that platelet PN-1 inhibited both the generation of plasmin by fibrin-bound tissue plasminogen activator and the activity of fibrin-bound plasmin itself. Rotational thromboelastometry and laser scanning confocal microscopy were used to demonstrate that PN-1 blockade or deficiency resulted in increased clot lysis and in an acceleration of the lysis front. Protease nexin-1 is thus a major determinant of the lysis resistance of platelet-rich clots. Moreover, in an original murine model in which thrombolysis induced by tissue plasminogen activator can be measured directly in situ, we observed that vascular recanalization was significantly increased in PN-1-deficient mice. Surprisingly, general physical health, after tissue plasminogen activator-induced thrombolysis, was much better in PN-1-deficient than in wild-type mice. Our results reveal that platelet PN-1 can be considered as a new important regulator of thrombolysis in vivo. Inhibition of PN-1 is thus predicted to promote endogenous and exogenous tissue plasminogen activator-mediated fibrinolysis and may enhance the therapeutic efficacy of thrombolytic agents.

Endogenous Tissue Plasminogen Activator Mediates Bone Marrow Stromal Cell-Induced Neurite Remodeling After Stroke in Mice

Bone marrow stromal cells (BMSC) decrease neurological deficits in rodents after stroke and concomitantly induce extensive neurite remodeling in the brain, which highly correlates with the improvement of neurological function. We investigated the effects of endogenous tissue plasminogen activator (tPA) on neurite remodeling after BMSC treatment. Adult C57BL/6 wild-type (WT) mice and tPA knockout (tPA(-/-)) mice were subjected to middle cerebral artery occlusion, followed by an injection of 1×10(6) BMSC (n=18) or phosphate-buffered saline (n=18) into the tail vein 24 hours later. Behavioral tests were performed at 3, 7, and 14 days after middle cerebral artery occlusion. Animals were euthanized at 14 days after stroke. The effects of BMSC on functional recovery depended on presence or absence of tPA, even after adjusting for imbalanced stroke severity. BMSC significantly improve functional recovery in WT mice compared to WT controls but show no beneficial effect in the tPA(-/-) mice compared to tPA(-/-) controls. Axonal density and synaptophysin-positive areas along the ischemic boundary zone of the cortex and striatum in WT mice are significantly higher than in the tPA(-/-) mice. BMSC treatment significantly increases tPA protein level and activity only in WT mice. Our results suggest that endogenous tPA promotes BMSC-induced neurite outgrowth and may contribute to functional recovery after stroke.

The Kringle-2 domain of tissue plasminogen activator significantly reduces mortality and brain infarction in middle cerebral artery occlusion rats

Tissue plasminogen activator (TPA) showed brain-protective activity within the first 15 min after cerebral ischemia in rats. To understand its molecular mechanism, TPA derivates were intracerebroventricularly administered at 15 min before, and 15, 90, 120 min after middle cerebral artery occlusion (MCAO) in rats. The reduction in mortality and cerebral infarction at 24 h was seen only with TPA administered at 15 min after MCAO. The down-regulation of endogenous TPA by the intracerebroventricular injection of TPA was found to be responsible for the protective effect on the integrity of blood-brain barrier after MCAO, as well as for the reduction in mortality and cerebral infarction. Moreover, for the first time we have found that the Kringle-2 domain is essential for the brain-protective activity of TPA.

Endogenous tissue plasminogen activator increases hemorrhagic transformation induced by heparin after ischemia reperfusion in rat brains

Objective: Tissue plasminogen activator (tPA) as a main thrombolytic drug for acute ischemic stroke remains complicated by risk of hemorrhagic transformation. However, whether endogenous tPA is also involved in hemorrhagic transformation is yet unclear.Methods: We randomly assigned male Sprague–Dawley rats into three groups: the heparin group, the control group and the sham operated group. The ischemic rat models were induced by middle cerebral artery occlusion through intraluminal thread technique for 2 hours, followed by 24 hours of reperfusion. Heparin or saline was intermittent peritoneally injected after reperfusion. The extent of cerebral hemorrhage, the infarct volume, as well as the content and activity of endogenous tPA were evaluated. The matrix metalloproteinase 9 (MMP-9) expression and activity were also measured.Results: All rats receiving heparin after reperfusion were subjected to hemorrhagic transformation. Hemorrhage volume in the heparin group was remarkably present. There was significant difference between the two groups (p<0·01). In the heparin group, the expressions of endogenous tPA and MMP-9 obviously increased, while their content and activity had significant differences compared with that of the control group (p<0·01).Conclusion: Endogenous tPA, through enhancement of MMP-9 expression and proteolytic activation, plays an important role in the pathogenesis of hemorrhagic transformation after cerebral reperfusion induced by heparin.