Elevated Plasma Tissue-type Plasminogen Activator (t-PA) and Soluble Throm-bomodulin in Patients Suffering From Severe Acute Respiratory Syndrome (SARS) as a Possible Index for Prognosis and Treatment Strategy

To detect the presence of endothelial injury in patients with severe acute respiratory syndrome （SARS） via enhanced levels of tissue-type plasminogen activator （t-PA） and soluble thrombomodulin （sTM）. Methods Case patients were from Xuanwu Hospital （Capital University of Medical Sciences, Beijing, China）, and all of them met clinical criteria for SARS. Healthy controls were some of the hospital employees. Endothelial injury bio-markers tPA and sTM were detected by commercial ELISA-methods. Results Classic plasma markers of endothelial injury, tPA and sTM significantly elevated in SARS patients in comparison to controls [t-PA： 1.48±0.16 nmol/L versus 0.25±0.03 nmol/L （P〈0.0001）, and sTM： 0.26±0.06 nmol/L versus 0.14±0.02 nmol/L （P〈0.05）]. The only patient who died had extremely high levels of these endothelial injury markers （t-PA： 2.77 nmol/L and sTM： 1.01 nmol/L）. The likelihood ratio analysis indicated the excellent discriminating power for SARS at the optimal cut-point of 0.49 nmol/L for tPA and 0.20 nmol/L for sTM, respectively. Significant numerical correlations were found among these endothelial injury markers in SARS patients. The numerical coefficient of correlation Pearson r between t-PA and sTM was 0.5867 （P〈0.05）. Conclusion Increased plasma concentrations of tPA and sTM in patients with SARS suggest the possibility of endothelial injury. SARS patients might need anticoagulant therapy or fibrinolytic therapy in order to reverse intraalveolar coagulation, microthrombi formation, alveolar and interstitial fibrin deposition. It may not only provide a useful treatment and prognostic index but also allow a further understanding of the pathological condition of the disease.

Intraclot Recombinant Tissue-type Plasminogen Activator Reduces Perihematomal Edema and Mortality in Patients with Spontaneous Intracerebral Hemorrhage

The study aimed to investigate the impact of intraclot recombinant tissue-type plasminogen activator （rt-PA） on perihematomal edema （PHE） development in patients with intracerebral hemorrhage （ICH） treated with minimally invasive surgery （MIS） and the effects of intraclot rt-PA on the 30-day survival. We reviewed the medical records of ICH patients undergoing MIS between October 2011 and July 2013. A volumetric analysis was done to assess the change in PHE and ICH volumes at pre-MIS （T1）, post-MIS （T2） and day 10-16 （T3） following diagnostic computed tomographic scans （To）. Forty-three patients aged 52.8±11.1 years with （n=30） or without rt-PA （n=13） were enrolled from our institutional ICH database. The median rt-PA dose was 1.5 （1） mg, with a maximum dose of 4.0 mg. The ratio of clot evacuation was significantly increased by intraclot rt-PA as compared with controls （77.9%±20.4% vs. 64%±15%; P=0.046）. From TI to T2, reduction in PHE volume was strongly associ- ated with the percentage of clot evacuation （p=0.34; P=-0.027）. In addition, PHE volume was positively correlated with residual ICH volume at the same day （p ranging from 0.39-0.56, P〈0.01）. There was no correlation between the cumulative dose of rt-PA and early （T2） PHE volume （p=0.24; P=0.12） or de- layed （T3） PHE volume （p=0.19; P=0.16）. The 30-day mortality was zero in this cohort. In the selected cohort of ICH patients treated with MIS, intraclot rt-PA accelerated clot removal and had no effects on PHE formation. MIS aspiration and low dose of rt-PA seemed to be feasible to reduce the 30-day mor- tality in patients with severe ICH. A large, randomized study addressing dose titration and long-term outcome is needed.

Activity of Ginkgo biloba Extract and Quercetin on Thrombomodulin Expression and Tissue-type Plasminogen Activator Secretion by Human Umbilical Vein Endothelial Cells

Objective In order to investigate the pharmacological properties of Ginkgo biloba extract （GBE） on improving blood circulation, the regulating action of GBE and quercetin （a main flavonoid ingredient in GBE） on thrombomodulin （TM） expression and tissue-type plasminogen activator （t-PA） secretion was studied. Methods Using flow cytometer and gel image system respectively, we evaluated the TM expression and the t-PA secretion by human umbilical vein endothelial cells （HUVECs） in vitro. Results The increase of TM expression on HUVECs surface was induced by GBE rather than quercetin in a dose- and time-dependent manner. Both GBE and quercetin increased the t-PA release significantly. Conclusion The effect of GBE on improving blood circulation may be partly attributed to its promoting TM expression and t-PA secretion by endothelial ceils, and quercetin participated in the effect of GBE on t-PA secretion. However, the action of GBE on increasing TM expression needs further study.

Experimental Study of Tissue-type Plasminogen Activator Gene to Prevent Vein Grafts Stenosis

The effects of in vivo local expression of recombined human tissue-type plasminogen activator （t-PA） gene on the thrombosis and neointima formation of vein grafts were explored. Jugular vein-to-artery bypass grafting was performed on 72 New Zealand white rabbits. The rabbits were divided into 3 groups according to the different processing methods： transfected t-PA gene group （n = 24）, vector group （n= 24） and blank control group （n = 24）. Samples of vein grafts were harvested at different time points after surgery. The expression of t-PA gene in vein graft was detected by RT-PCR and the synthesis of t-PA protein by Western-Blot assay. The t-PA activity was measured by chromogenic substrate assay. The Cr51 labeled platelets accumulation in vein grafts was counted. The histopathological changes were compared in intima hyperplasia index among the three groups after operation. The results showed that at the 2^nd , 5^th , 14^th and 28^th day after operation, RT-PCR and Western-blot confirmed the expression of t-PA mRNA and protein at the site of gene transfer. The t-PA activity detected on the 2^nd, 5^th, 14^th and 28^th day in experimental group was 370. 63±59. 44, 344. 13±48. 47, 252.87±51.80 and 161.75±68. 94 U/g respectively, and disappeared on the 60^th day and undetected in the control groups. The number of platelets accumulated in the vein grafts in gene group, vector group and blank control group was （85. 04 ± 21.58） 10s, （225.87±85.13） 10^6 and （211.57±78.02） 10^6 respectively. The number of platelets accumulated in gene group was significantly fewer than that in the control groups. Morphometric analysis revealed that intimal hyperplasia was markedly reduced in the t-PA gene group as compared with that in the control groups. It was suggested that the local expression of t-PA gene in vein graft significantly inhibited the accumulation of platelets, thrombosis and concomitant intimal hyperplasia, by which stenosis of bypass graft could be prevented effectively.

Tissue-type plasminogen activator is a homeostatic regulator of synaptic function in the central nervous system

Membrane depolarization induces the release of the serine proteinase tissue-type plasminogen activator（t PA） from the presynaptic terminal of cerebral cortical neurons.Once in the synaptic cleft this t PA promotes the exocytosis and subsequent endocytic retrieval of glutamate-containing synaptic vesicles,and regulates the postsynaptic response to the presynaptic release of glutamate.Indeed,t PA has a bidirectional effect on the composition of the postsynaptic density（PSD） that does not require plasmin generation or the presynaptic release of glutamate,but varies according to the baseline level of neuronal activity.Hence,in inactive neurons t PA induces phosphorylation and accumulation in the PSD of the Ca^（2＋）/calmodulin-dependent protein kinase IIα（pCa MKIIα）,followed by pCa MKIIα-induced phosphorylation and synaptic recruitment of Glu R1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid（AMPA） receptors.In contrast,in active neurons with increased levels of pCa MKIIα in the PSD t PA induces pCa MKIIα and p Glu R1 dephosphorylation and their subsequent removal from the PSD.These effects require active synaptic N-methyl-D-aspartate（NMDA） receptors and cyclin-dependent kinase 5（Cdk5）-induced phosphorylation of the protein phosphatase 1（PP1） at T320.These data indicate that t PA is a homeostatic regulator of the postsynaptic response of cerebral cortical neurons to the presynaptic release of glutamate via bidirectional regulation of the pCa MKIIα/PP1 switch in the PSD.

Tissue plasminogen activator-independent roles of neuroserpin in the central nervous system

A number of studies have confirmed the existence of tissue-type plasminogen activator-independent roles of neuroserpin, a member of the serine protease inhibitor superfamily. In this review article, we aim to clarify this role. These unique roles of neuroserpin are involved in its neuroprotective effect during ischemic brain injury, its regulation of tumorigenesis, and the mediation of emotion and cognition through the inhibition of urokinase-type plasminogen activator and fibrinolysin, modification of Th cells, reducing plaque formation, promoting process growth and intracellular adhesion, and alterina the expression of cadherin and nuclear factor kaooa B.

The plasminogen activating system in the pathogenesis of Alzheimer’s disease

Dementia is a clinical syndrome that affects approximately 47 million people worldwide and is characterized by progressive and irreversible decline of cognitive,behavioral and sesorimotor functions.Alzheimer’s disease(AD)accounts for approximately 60–80%of all cases of dementia,and neuropathologically is characterized by extracellular deposits of insoluble amyloid-β(Aβ)and intracellular aggregates of hyperphosphorylated tau.Significantly,although for a long time it was believed that the extracellular accumulation of Aβwas the culprit of the symptoms observed in these patients,more recent studies have shown that cognitive decline in people suffering this disease is associated with soluble Aβ-induced synaptic dysfunction instead of the formation of insoluble Aβ-containing extracellular plaques.These observations are translationally relevant because soluble Aβ-induced synaptic dysfunction is an early event in AD that precedes neuronal death,and thus is amenable to therapeutic interventions to prevent cognitive decline before the progression to irreversible brain damage.The plasminogen activating(PA)system is an enzymatic cascade that triggers the degradation of fibrin by catalyzing the conversion of plasminogen into plasmin via two serine proteinases:tissue-type plasminogen activator(tPA)and urokinase-type plasminogen activator(uPA).Experimental evidence reported over the last three decades has shown that tPA and uPA play a role in the pathogenesis of AD.However,these studies have focused on the ability of these plasminogen activators to trigger plasmin-induced cleavage of insoluble Aβ-containing extracellular plaques.In contrast,recent evidence indicates that activity-dependent release of uPA from the presynaptic terminal of cerebral cortical neurons protects the synapse from the deleterious effects of soluble Aβvia a mechanism that does not require plasmin generation or the cleavage of Aβfibrils.Below we discuss the role of the PA system in the pathogenesis of AD and the translational relevance of data published to this date.

Effects of simvastatin on cigarette smoke extract induced tissue-type plasminogen activator and plasminogen activator inhibitor-1 expression in human umbilical vein endothelial cells

Background Cigarette smoking has an influence on both arterial-type and venous-type thrombosis. However, little is known about the direct effect of cigarette smoke extract （CSE） on fibrinolytic activity of human umbilical vein endothelial cells （HUVECs）. Most recently, simvastatin has been marked in its effect on endothelial cells protection and anticoagulation. In this study, the effect of CSE on the expression of tissue-type plasminogen activator （t-PA） and plasminogen activator inhibitor-l（PAl-1） in HUVECs was addressed. The role of simvastatin in CSE-induced fibrinolytic activity changes was investigated as well. Methods The fourth to fifth generation of HUVECs were incubated respectively with 0, 5%, 10% and 20% CSE for 6 hours or exposed to 5% CSE for 0, 4, 6, 8, 12, 24 hours to determine the expression changes of t-PA and PAl-1 protein. Meanwhile, cells were also accordingly exposed either to 5% CSE alone or simvastatin pre-treated and 5% CSE for 24 hours to assess the role of simvastatin in CSE-induced t-PA and PAl-1 protein and mRNA expression in HUVECs. RT-PCR and ELISA techniques were used for detecting the t-PA or PAl-1 mRNA and protein. Results After 6-hour exposure to CSE, the expression levels of t-PA protein in 10% and 20% CSE-treated groups reduced significantly （（0.0365±0.0083） ng/ml, （0.0255±0.0087） ng/ml） when compared with that of control group （（0.0660±0.0120） ng/ml） （P 〈0.05）. In contrast, the levels of PAl-1 protein in 5%, 10% and 20% CSE-treated groups increased remarkably （（13.3225±0.5680） ng/ml, （14.2675±1.5380） ng/ml, （14.4292±1.6230） ng/ml） when compared with that of control group （（8.5193_±0.7537）ng/ml） （P〈0.05）. After stimulation with 5% CSE for 0, 4, 6, 8, 12, 24 hours, the levels of PAl-1 protein increased over time and reached the peak at 24 hours （（14.6400±1.0651） ng/ml）, which was significantly higher than that of control group （（12.0656±0.6148） ng/ml） （P 〈0.05）. Additionally, CSE could up-regulate PAl-1 expression at both the mRNA and the protein levels. The levels of PAl-1 mRNA and protein increased significantly in 5% CSE-treated group （（8.8030±0.4745） ng/ml, （1.8155±0.0412） ng/ml） compared with those of control groups （（5.0588±0.2315） ng/ml, （1.3030±0.0647） ng/ml） （P 〈0.01）, and decreased after 2-hour simvastatin pre-treatment （（5.4875±0.3166） ng/ml, （1.3975-±0.0297） ng/ml） （P 〈0.01）. No significant difference was found at the levels of t-PA protein and mRNA （P 〉0.05）. Conclusions CSE inhibits the fibrinolytic activity of HUVECs in vitro. Simvastatin plays a protective role in CSE-induced fibrinolytic malfunction.

Identification and Possible Function of Tissue-type and Urokinase-type Plasminogen Activators and Plasminogen Activator Inhibitor in Corpus Luteum of Rhesus Monkey

Two types of plasminogen activators, tissue type (tPA) and urokinase type(uPA),and a plasminogen activator inhibitor (PAI-1) have been identified in ovariangranulosa cells (GC) of both rat and rhesus monkey. Interaction and coordinated expression of tPA and PAI-1 in the same tissue cells are closely related with certain reproductive processes such as ovulation, spermatogenesis and endometrium cycle. In our previous studies on gonadotropin-induced ovulation in rhesus monkeys,we

Construction,expression and characterization of tissue-type plasminogen activator mutants

Three tissue-type plasminogen activator(t-PA)mutants were constructed by recombinant andsite-directed mutagenesis techniques.They are del(296—302)with deletion of PAI-1 binding site,N117Q/N184Qwith deglycosylation of K1 and K2 domains,and their combination mutant designated as GGI.Then these threemutants were suocessfully transiently expressed in COS-7 ceils,and GGI was further stably expressed in CHOcells.The biological characterization of the expression products indicated that del(296—302)and GGIpossessed the resistance to inhibition by PAI-1.In addition,the specific activity of GGI was increased byabout 46,the plasma half-life was prolonged by about one fold,while its affinity for fibrin was not affected.

CLINICAL STUDY OF T-PA AND U-PA EXPRESSION IN PATIENTS WITH GASTROINTESTINAL CANCER

Objective: To study the changes of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) expressions and fibrinolysis molecular markers in patients with gastrointestinal cancer in order to elucidate their clinical significance. Methods: The plasma levels of t-PA, u-PA, urokinase-type plasminogen activator receptor (u-PAR) and plasmin anti-plasmin complex (PAP) were measured by ELISA. t-PA and u-PA mRNAs were detected by Real-time RT-PCR. Results: The plasma levels of u-PA, u-PAR and PAP were elevated in gastrointestinal cancer patients, while u-PA was markedly elevated in patients with local infiltration, lymph node involvement or distal metastasis. U-PA mRNA was higher and t-PA was lower in gastrointestinal cancer compared to normal tissue. Conclusion: Hyperfibrinolysis is an important factor related with metastasis potential of gastrointestinal cancer. t-PA may be a character of well differentiated tissue.

Efficacy of Sanqi(Radix Notoginseng)in treating cerebral hemorrhage in rats with traumatic brain injury

OBJECTIVE:To evaluate the protective efficacy of Sanqi(Radix Notoginseng)on cerebral hemorrhage in a rat model of traumatic brain injury(TBI)by investigating plasminogen activator inhibitor-1(PAI-1),tissue-type plasminogen activator(t-PA),nuclear factor-κB(NF-κB,p-p65),nitric oxide(NO),endothelin(ET),cluster differentiation(CD61CD62),and coagulation.METHODS:The free-fall method was used to create a rat model of TBI.Forty-eight rats were randomly divided into six groups:the blank group,sham group,model group,low-dose Sanqi(Radix Notoginseng)group,middle-dose Sanqi(Radix Notoginseng)group,and high-dose Sanqi(Radix Notoginseng)group.At 24 h after the model was created,we investigated brain MRI,brain tissue morphology using HE staining,flow cytometry,and immunohistochemical changes.RESULTS:Cerebral hemorrhage was aggravated in TBI rats(observed in brain specimens,brain MRI,and brain tissue HE).Cerebral immunohistochemistry results demonstrated that the expression of t-PA,PAI-1 and p-p65 increased significantly in TBI rats,while t-PA/PAI-1 had a significant decrease.In addition,CD61CD62,D2D,and ET were significantly increased in TBI rats,and PT and APTT were significantly prolonged;in contrast,NO was significantly decreased.Sanqi(Radix Notoginseng)decreased cerebral hemorrhage in TBI rats(observed in brain MRI and brain tissue HE),and increased t-PA/PAI-1,CD61CD62 significantly.It also significantly decreased the expression of t-PA,PAI-1,and p-p65 in brain immunohistochemistry and significantly decreased PT,APTT,D2D,and ET.However,there were no differences in NO between the model group and the Sanqi(Radix Notoginseng)group.CONCLUSION:Sanqi(Radix Notoginseng)can decrease the expression of p-p65,increase t-PA/PAI-1,and stem traumatic intracranial hemorrhage in a TBI rat model.

Effects of Propyl Gallate on Carotid Artery Thrombosis and Coagulation/Fibrinolysis System in Rats

Objective： To investigate the effects of propyl gallate （PrG） on the thrombus formation time and the coagulation/fibrinolysis system in an experimental carotid artery thrombosis model in rats. Methods： Fifty SD rats were randomly divided into 5 groups （10 animals/group）： the normal group （normal saline 2 mL/kg）, the model group （normal saline, 2 mL/kg）, the heparin control group （1 250 IU/kg）, the low dose PrG group （30 mg/kg）, and the high dose PrG group （60 mg/kg）. Thirty minutes after intravenous injection of saline or the corresponding drugs, a carotid artery thrombus was induced by continuous electric stimulation in all rats except for those in the normal group. The duration from the initiation of the electric stimulation to the sudden drop in carotid temperature was recorded as the thrornbus formation time. Levels of plasma tissue-type plasminogen activator （t-PA） and plasminogen activator inhibitor-1 （PAl-1） were determined by ELISA. Results： PrG （30 and 60 mg/kg） can prolong the thrombus formation time, but the effect was obviously weaker than that of heparin （P〈0.05, P〈0.01）. Compared with the model group, PrG （30 and 60 mg/kg） elevated the plasma activity of t-PA （both P〈0.05） and showed an increasing tendency in elevating the ratio of t-PA/PAI-1 （P〉0.05）, while it had no significant effect on the level of PAI-1. Conclusion： PrG has a certain antithrombotic effect and can slightly regulate the imbalance of the t-PA/PAl-1 ratio.

An Experimental Study on the Flexibility of Prevention against Thrombosis Following Mechanical Valve Replacement by tPA Gene Transduction

Objectives Use a gene suture immersed recombinant tissue-type plasminogen activator （r-tPA）expression plasmid to transduce myocardia to prevent the thrombosis after mechanical tricuspid valve replacement in pigs. Methods A r-tPA gene plasmid was constructed and conjugated to a novel cationic phosphonolipid and a r-tPA gene suture was made. Eighteen pigs were selected and divided into two groups at randomization. There were 9 pigs in the experimental group and 9 in the control group, all the 18 pigs＇ tricuspids were replaced with mechanical valves. The gene threads were sutured into the right ventricular walls near mechanical valves and an ultrasound was used on the surfaces of the right ventricular walls for the gene transfer in the experimental group. Coagulative function, D-dimer level of the blood and the thrombosis on the surfaces of the valves were observed. Results r-tPA gene plasmid was successfully constructed and r-tPA protein was expressed in the ventricular cells around the gene sutures. D-dimer reached its peak level （ 1.67 ±0. 79） μg · mL^-1 in 1 week after operation in two groups, but it decreased to preoperation level thereafter in control group and kept on the high level and reincreased to a new high level （ 1.89 ± 0.79 ） μg · mL^-1 until the end of the experiment in experimental group. The thromboses around the valves were found in all the control group （100%） but only 1 （ 11.11% ） case in experimental group. There were no changes in prothrombin time pre and post operation in two groups. Conclusions Using gene suture immersed r-tPA expression plasmid to transduce myocardia might be a best substitution for life long anti-coagulation therapy for the patients, who underwent operation.