Relationships between euglobulin clot lysis time and the plasma levels of tissue plasminogen activator and plasminogen activator inhibitor 1

The relationships between tissue plasminogen activator (tPA), its fast acting inhibitor (PAI-1) and euglobulin clot lysis time (ELT) were investigated with healthy volunteers' plasma. Turbidimetric clot lysis assay by the microtiter plate reader was utilized for ELT with a slight modification. Both tPA and PAI-1 showed the significant correlation with ELT. tPA had a significantly positive, not negative, correlation with ELT (R = 0.387, p less than 0.001). Higher correlation coefficients (R = 0.580, p less than 0.001 and R = 0.599, p less than 0.001) were obtained between ELT and total PAI-1 or free PAI-1 than tPA or tPA-PAI-1 complex (R = 0.427, p less than 0.001). The positive correlation was also obtained between tPA and PAI-1. These data suggest that PAI-1 is a highly important factor for ELT, especially, the amounts of free PAI-1 being the key factor to determine the ELT, which can represent the potential activity of the fibrinolytic system.     

Tissue plasminogen activator, plasminogen activator inhibitor-1, and tissue plasminogen activator/plasminogen activator inhibitor-1 complex as risk factors for the development of a first stroke

BACKGROUND NAD PURPOSE: Abnormalities in the fibrinolytic system have been associated with an increased risk for stroke in a few studies. This study was designed to test whether plasma levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and tPA/PAI-1 complex could predict a first-ever stroke. METHODS: The study was an incident case-control study nested within the V?sterbotten Intervention Program and the Northern Sweden Monitoring Trends and Determinants in Cardiovascular Disease (MONICA) cohorts. In this study 108 first-ever stroke cases were defined according to the MONICA classification, and 216 controls from the same cohort were randomly selected and matched for age, sex, sampling time, and geographic region. RESULTS: Stroke occurred on average 30 months after the blood sampling date. The mean plasma concentration of tPA/PAI-1 complex was higher for the stroke cases than for the controls (3.9 versus 3.0 microgram/L). In univariate regression analysis, significantly higher odds ratios were found for the tPA/PAI-1 complex as continuous variable. When divided into quartiles, the odds ratio was 2.74 for the highest quartile compared with the lowest. In the multivariate model, the tPA/PAI-1 complex remained an independent predictor for stroke. Additionally, tPA mass concentration quartiles 3 and 4 showed a significant association with all stroke as outcome. No association was found, however, for PAI-1. In subgroup analysis of cerebral hemorrhage (n=18), the mean tPA/PAI-1 complex level was higher for the cases than for the controls (4.8 versus 3.0 microgram/L), and in multivariate analysis including all controls (n=216), only tPA/PAI-1 complex remained significant. CONCLUSIONS: This prospective study shows that tPA/PAI-1 complex, a novel fibrinolytic marker, is independently associated with the development of a first-ever stroke, especially hemorrhagic stroke. This finding supports the hypothesis that disturbances in fibrinolysis precede a cerebrovascular event.     

Enhanced lytic efficacy of multiple bolus injections of tissue plasminogen activator in dogs

The purpose of this study was to compare the lytic efficacy of tissue plasminogen activator (tPA) following different dosing regimens. Radiolabelled clots from dog whole blood were inserted into an extracorporeal jugular loop in anesthetized dogs. Clot size (counts/min) was continuously recorded. tPA was administered as a single 1 min bolus of 0.4 mg/kg, as four multiple bolus injections of 0.1 mg/kg each at 30 min intervals, or as an initial bolus (0.04 mg/kg) followed by a 30 min infusion of 0.36 mg/kg (10%/90%, bolus/infusion). Multiple injections of the same total dose of tPA resulted in 76% and 51% greater clot lysis than single bolus injection or bolus/infusion regimen, respectively, measured 120 min after initial dosing. There were no differences in fibrinogen, plasminogen or alpha-2-antiplasmin between the different treatment groups at 120 min.     

The effect of insulin treatment on the balance between tissue plasminogen activator and plasminogen activator inhibitor-1 in type 2 diabetic patients.

Beside hypercoagulation and hyperactivated platelets disturbances of the fibrinolytic system towards hypofibrinolysis have been reported to be associated with both glycemic and lipidemic derangement in diabetic patients. In the present prospective follow-up study the effect of 16 weeks insulin treatment and glycemic regulation on plasma levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), the main regulators of fibrinolysis, was investigated in 19 type-2 diabetic patients with secondary failure to sulphonylureas. A similar glycemic regulation was obtained in a control group of 10 type 2 diabetic patients with sufficient metabolic response to strict dietary treatment and continuation of sulphonylurea treatment. Compared to 27 healthy subjects levels of tPA and PAI-1 were not significantly increased in type 2 diabetic patients before metabolic intervention. Although a hypofibrinolytic state due to an increase of PAI-1 levels was previously reported in obese hyperinsulinemic patients, no effect of insulin treatment on both tPA- and PAI-1 levels was observed in the present study including patients with only slightly increased body mass index (median 26.0 kg/m2). By correlation analysis PAI-1 levels were significantly related to serum cholesterol (R = 0.52) and glycemic control (glucose R = 0.41) in the whole group of diabetic patients at entry and in both subgroups after 16 weeks of treatment (insulin group: cholesterol R = 0.46, HbA1c R = 0.51; sulphonylurea group: cholesterol R = 0.59, HbA1c R = 0.58). In healthy subjects tPA and PAI-1 was correlated to serum insulin (R = 0.54, R = 0.56) and triglycerides (R = 0.46, R = 0.40).(ABSTRACT TRUNCATED AT 250 WORDS)     

Vampire bat salivary plasminogen activator promotes robust lysis of plasma clots in a plasma milieu without causing fluid phase plasminogen activation.

Vampire bat salivary plasminogen activator (BatPA), human tissue-type plasminogen activator (tPA) or streptokinase (SK) were incubated in human citrated plasma containing a plasma clot that was radiolabelled with iodine-125 fibrin(ogen). Complete clot dissolution by BatPA (30 nM) was associated with slight activation of "fluid phase" plasminogen; the plasma levels of functional fibrinogen and alpha 2-antiplasmin decreased by only 8 and 19%, respectively. Addition of SK (3,600 IU/ml) to the clot-containing plasma caused complete clot lysis and massive activation of the "fluid phase" plasminogen, leading to greater than 60 and 96% decreases of the functional levels of fibrinogen and alpha 2-antiplasmin, respectively. Incubation of tPA (30 nM) in clot-containing plasma caused complete clot lysis as well as substantial activation of "fluid phase" plasminogen; the plasma levels of functional fibrinogen and alpha 2-antiplasmin decreased by 45 and 79%, respectively. The profound degradation of fibrinogen in the SK and tPA but not BatPA-containing samples was confirmed by immunoblot analysis. Additional experiments showed that the presence of soluble clot lysate in plasma containing tPA enhanced the extent of fibrinogen degradation from 25% to greater than 60%; the addition of soluble clot lysate to the plasma containing BatPA did not prompt further fibrinogen degradation. Finally, studies using exogenous alpha 2-antiplasmin suggested that plasmin generated via tPA-mediated activation of "fluid phase" plasminogen does not play an important role in clot dissolution.     

Plasminogen activator inhibitor type 2 inhibits cell surface associated tissue plasminogen activator in vitro: potential receptor interactions

Regulation of cellular plasminogen activation is necessary for maintenance of tissue homeostasis. Despite increasing evidence for co-expression of tissue type plasminogen activator (tPA) and plasminogen activator inhibitor type-2 (PAI-2; SERPINB2) under patho/physiological conditions, the inhibition of cell-bound tPA mediated plasminogen activation by PAI-2 has not been addressed. Here we show that PAI-2 can inhibit cell-bound tPA activity in vitro and thus prevent plasmin formation. We also examined the potential involvement in this inhibition of the annexin II heterotetramer (AIIt), one of the many well characterized cell-surface co/receptors for tPA and plasminogen that efficiently promotes plasminogen activation. This receptor was of interest because AIIt has also been shown to directly bind PAI-2. Characterization of these potential interactions using purified protein systems revealed that PAI-2 directly bound AIIt via the p11 (S100A10) subunit. However, PAI-2 prevented AIIt/tPA-mediated plasminogen activation by its classic serpin inhibitory activity rather than through competition with tPA/plasminogen for binding. Further analysis showed that PAI-2 inhibited cell bound tPA-induced plasmin activity in both an AIIt-dependent and -independent manner. These data open new possibilities for further investigations regarding the regulation of cellular plasmin generation in vivo, especially in tissues where PAI-2 and tPA may be co-expressed.     

The potentiating effect of platelet on plasminogen activation by tissue plasminogen activator

A new role for platelets in fibrinolysis is proposed. Platelets (euglobulin from platelet rich plasma and from human platelet extract) may potentiate plasminogen activation by tissue plasminogen activator(tPA). The potentiating activity was detected by both chromogenic substrate and fibrin plate analysis. The fibrinolysispotentiating substance in the platelets required the presence of both tPA and plasminogen, suggesting that it potentiates the activation of plasminogen by tPA. This substance was not related to fibrinogen degradation products because it was also present in platelets from two afibrinogenemic patients and did not lose its activity when separated from fibrinogen-related antigen by Sepharose 2B gel filtration. Since platelets contain both activator(s) and inhibitor(s) of plasminogen activation by tPA, a balance between activator(s) and inhibitor(s) in platelets may also be required for control of the fibrinolytic pathway.     

血浆中PAI-1和TAFI水平与溶栓后出血性转化相关性研究

目的探索PAI及TAFI对急性脑梗死静脉溶栓后出血性转化的预警作用。方法前瞻性的将溶栓患者分为出血转化组和无出血转化组,对两组患者溶栓前及溶栓后静脉血浆PAI及TAFI浓度进行对比及统计学分析。结果溶栓前两组基线PAI-1水平差异有明显统计学意义(P=0.037)。溶栓后次日复查晨血PAI-1水平出血转化组明显较无出血转化组值更低(P=0.035)。溶栓前出血转化组的基线TAFI水平较无出血转化组比较TAFI值更低(P=0.024)。溶栓后次日复查晨血出血转化组TAFI值同样低于无出血转化组(P=0.042)。结论血浆中PAI-1和TAFI水平与溶栓后出血性转化相关性较高。当溶栓前及溶栓后PAI-1及TAFI的低水平表达均可增加溶栓后出血性转化的风险。     

Plasma levels of free tissue factor pathway inhibitor in patients with various thyroid disorders

Various coagulation abnormalities occur in thyroid disorders and its range may vary from subclinical laboratory abnormalities to clinically significant disorders of coagulation. Tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of tissue factor mediated coagulation pathway, is reported to be increased in patients with Graves disease (GD) in one study. Hyperthyroid (n=10), hypothyroid (n=10) and subclinical hypothyroid (n=10) patients and control cases (n=16) were evaluated for free and total tissue factor pathway inhibitor (tTFPI), plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) levels in disease and euthyroid states. Free TFPI levels were significantly higher in hyperthyroid patients compared with the control group and subclinic hypothyroid patients (p<0.001), but not with hypothyroid patients (p>0.05). After the euthyroid state was obtained in the hyperthyroid group, the levels of total TFPI (p<0.05), free TFPI (fTFPI) (p<0.005), t-PA (p<0.005) and PAI-1 (p<0.02) decreased significantly. In hyperthyroid patients, there was a strong correlation between thyroid functions and free TFPI levels. In conclusion, we hypothesize that coagulation abnormalities seen in thyroid disorders cannot be explained directly with the impaired fibrinolytic activity but also with the elevated fTFPI levels. Both increased plasma fTFPI and PAI-1 levels could be markers of the peripheral activity of thyroid hormones.     

A new sensitive chromogenic substrate assay of tissue factor pathway inhibitor type 1

The present assay is a modification of our previously published two-stage chromogenic substrate assay of tissue factor pathway inhibitor type-1 (TFPI) activity [1]. In the first stage, the reaction mixture was made with factor VIIa (FVIIa) molecules in excess of tissue factor (TF) binding sites and contained diluted plasma, recombinant FVIIa (10 nM), recombinant TF (1/400 vol/vol), bovine factor Xa (1,1 nM), I-2882(R) (100 microg/ml), and CaCl(2) (10 mM). The fibrin polymerisation inhibitor I-2882(R) was added to the reaction mixture to prevent formation of cross-linked fibrin. In the second stage, residual TF/FVIIa catalytic activity was measured by the addition of a substrate mixture that contained bovine factor X and a chromogenic substrate (S-2222(R)). Standard curves were constructed using serial dilutions (0-1%) of pooled normal plasma. The dose-response relationship for serial dilutions of plasma was linear. The intra-assay coefficient of variations (CVs) for pre- and postheparin plasma samples (i.e., normal and high TFPI levels) were 1.7% and 9.9%, respectively; the inter-assay CVs were 10.0% and 19. 7%, respectively. The effect of variation in antithrombin activity on the assay was approximately 5%. The present assay correlated fairly well with our previously published assay (r=0.82, n=100) and with a commercial TFPI activity assay (Actichrome(R) TFPI Activity Assay, American Diagnostica, Greenwich, CT, USA; r=0.90, n=100), as well as with an antigen assay for TFPI total antigen (Imubind(R), American Diagnostica; r=0,96, n=100). Altman and Bland plots revealed that our previous assay underestimated TFPI activity at high TFPI levels (i.e., postheparin TFPI samples) compared with the other methods.     

Homocysteine and hemostatic disorder as a risk factor for myocardial infarction at a young age

Introduction: Hyperhomocysteinemia is a coronary risk factor, but its pathophysiologic mechanism remains unclear. Materials and methods: The importance of hyperhomocysteinemia in the pathogenesis of early myocardial infarction, was determined in case-control study of 127 men with a first early myocardial infarction ≤45 years and 150 age-matched male controls. We measured plasma concentrations of homocysteine, fibrinogen, antithrombin, tissue factor, tissue factor pathway inhibitor, tissue plasminogen activator, plasminogen activator inhibitor-I, plasminogen, ₂-antiplasmin, lipoprotein(a), protein C, protein S, factor VII, and activated factor VII. Results: Homocysteine concentrations were higher in patients with early myocardial infarction than in controls (11.2±5.3 and 8.3±5.0 μmol/l, respectively, P<0.001). Hyperhomocysteinemia was associated with early myocardial infarction (odds RATIO=2.22, P<0.001) by multivariate logistic regression analysis. Tissue factor, antithrombin, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor-I, lipoprotein(a), diabetes, and smoking also had associations. In a stepwise logistic regression analysis, hyperhomocysteinemia was the strongest predictor of early myocardial infarction (R²=0.19, P<0.001). Hyperhomocysteinemia also had positive correlations with tissue factor (ρ=0.26, P=0.009), tissue factor pathway inhibitor (ρ=0.23, P=0.020), and tissue plasminogen activator (ρ=0.25, P=0.011) in patients with early myocardial infarction, but not in controls. Conclusions: Hyperhomocysteinemia is an independent risk factor for early myocardial infarction, and is associated with a hypercoagulable state mediated by the extrinsic coagulation cascade.     

Urokinase-type plasminogen activator and metalloproteinase-2 are independently related to the carotid atherosclerosis in haemodialysis patients

INTRODUCTION: Matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs) system, and fibrinolytic system, have been implicated as important factors in atherosclerosis and vascular remodeling. However, no data are yet available on the associations between these two systems in relation to carotid atherosclerosis in hemodialysis (HD) patients. MATERIAL AND METHODS: We compared plasma levels of MMP-2, MMP-9, TIMP-1, TIMP-2; the parameters of fibrinolytic system: tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPAR) and its soluble receptor (suPAR), plasminogen activator inhibitor-1 (PAI-1), plasmin-alpha2-antiplasmin (PAP) complexes; high sensitivity C-reactive protein (hs CRP) as a marker of inflammation and a surrogate of atherosclerotic disease-intima media thickness (IMT) in HD patients and in healthy controls. RESULTS: The values of the uPA, suPAR, PAP, MMP-2, TIMPs, hs CRP and IMT in the patients significantly exceeded those in controls. The concentrations of MMP-9, tPA and PAI-1 were similar in both investigated groups. uPA, uPAR and PAP were positively associated with MMP-2/TIMPs system; all mentioned above parameters (except TIMP-2) and hsCRP were associated with IMT. Multivariate analysis showed that uPA, MMP-2 and age were the strong independent variables linked to IMT values in HD patients. CONCLUSIONS: The patients on haemodialysis treatment have evidence of disordered fibrinolysis/proteolysis balance in the plasma, independently associated with IMT on multivariate analysis. These data suggest the importance of uPA and MMP-2 levels in the developing of atherosclerosis in these patients.     

Plasminogen and tissue plasminogen activator interact with antithrombin III

Human antithrombin III was demonstrated to bind plasminogen specifically in a time and concentration-dependent manner. The above binding was also confirmed using ligand western blot assays. The interaction of plasminogen was significantly (>90%) inhibited by lysine, indicating the involvement of kringles in binding antithrombin III. Plasminogen also bound to heparin-antithrombin III complex. In converse experiments, antithrombin III also interacted with immobilized plasminogen. Using carboxypeptidase B digestion, the plasminogen-binding site of antithrombin III was localized to the carboxy-terminus lysine of the anticoagulant protein. Tissue plasminogen activator also interacted with antithrombin III in a time- and concentration-dependent manner and its binding was also significantly (>90%) inhibited by lysine. Moreover, the interaction of plasminogen and tissue plasminogen activator with antithrombin III was competitive. These results provide the first evidence for the interaction of antithrombin III with fibrinolytic factors and suggest that antithrombin III may serve to localize these factors at the site of clot formation.     

Mice lacking tissue plasminogen activator and urokinase plasminogen activator genes show attenuated matrix metalloproteases activity after sciatic nerve crush

Plasminogen activators (PAs), tissue PA (tPA) and urokinase PA (uPA), have been shown to be induced in sensory neurons after sciatic nerve crush. These findings suggested that PAs facilitate peripheral nerve regeneration by digesting adhesive cell contacts and by activation of other proteases, thereby initiating a proteolytic cascade. Both tPA and uPA activate some matrix metalloproteases (MMPs), indirectly via plasminogen activation or directly, such as the uPA activation of MMP-2. In this study, we demonstrated, by using tPA and uPA knockout mice, that a lack of a plasminogen activator affected MMP-9 and MMP-2 activity after crushing of the sciatic nerve. These findings show that the PAs are important for MMP-9 and MMP-2 activity at the crush site.     

Tissue-type plasminogen activator -7,351C/T enhancer polymorphism is associated with a first myocardial infarction

We recently identified a polymorphic Sp1 binding site in an enhancer at the tissue-type plasminogen activator (tPA) locus (tPA -7,351C/T), which was associated with vascular tPA release. Subjects homozygous for the -7,351C allele had twice the tPA release rate compared to subjects carrying the -7,351T allele. In this study we tested the hypothesis that the tPA -7,351C/T polymorphism is associated with myocardial infarction (MI). In a population-based prospective nested case-control study within northern Sweden, genotypes were determined among 61 MI cases and 120 controls. In a multivariate model, the tPA -7,351C/T polymorphism (OR 2.68 for T allele carriers; 95% CI 1.31-5.50), tPA antigen (OR 1.16; 95% CI 1.07-1.25) and apo A-I (OR, 0.997; 95% CI 0.995-0.999) were independently associated with a first MI. These findings suggest that genetic markers of local tPA release and circulating steady-state tPA levels carry independent prognostic information.     

Uptake and degradation of tissue plasminogen activator in rat liver

The mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(alpha), and the terminal phase, t1/2 (beta), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells. Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells. Competitive inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types. These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.     

Modulation of zinc toxicity by tissue plasminogen activator

The tissue plasminogen activator (tPA)-plasmin proteolytic system mediates excitotoxin-induced neurodegeneration in vivo and in cell culture. tPA also confers neuroprotection from zinc toxicity in cell culture through a proteolysis-independent mechanism. This raises two questions: what is this non-enzymatic mechanism, and why tPA does not synergize with zinc to promote neuronal cell death? We show here that zinc binds to tPA and inhibits its activity in a dose-dependent fashion, thus terminating its protease-dependent neurotoxic capacity. We extend the previously reported culture findings to demonstrate that elevated zinc is neurotoxic in vivo, and even more so when tPA is absent. Thus, physiological levels of tPA confer protection from elevated free zinc. Mechanistically, tPA promotes movement of zinc into hippocampal neuron cells through voltage-sensitive Ca(2+) channels and Ca(2+)-permeable AMPA/KA channels. Therefore, zinc and tPA each appear to be able to limit the potential of the other to facilitate neurodegeneration, a reciprocal set of actions that may be critical in the hippocampus where tPA is secreted during the nonpathological conditions of learning and memory at sites known to be repositories of free and sequestered zinc.     

The 4G4G genotype of the plasminogen activator inhibitor 4G/5G gene polymorphism is associated with coronary atherosclerosis in patients at high risk for this disease.

BACKGROUND: Disturbances in fibrinolytic activity, such as increase in plasminogen activator inhibitor (PAI) activity, have been linked with an increased risk for coronary artery disease (CAD) and myocardial infarction (MI). Since 4G4G homozygotes of an insertion/deletion (4G/5G) gene variation in the promoter of PAI-I have been shown to have increased levels of PAI-I, we analysed the relation of this gene polymorphism to CAD and MI in a population of 2565 participants who underwent coronary angiography for diagnostic purposes. RESULTS: In the total sample, the PAI-I 4G/4G genotype was associated with the presence, but not with the extent of CAD. However, in a subgroup of former and present smokers (n = 1782) or of individuals with a BMI above the mean value of 26.9 kg x m(-2) (n = 1269), the PAI-I 4G4G genotype was not only associated with the presence, but also with the extent of CAD, defined either by the number of diseased vessels or by the CHD score according to Gensini. This observation also applied to other high-risk groups of individuals with high BMI and hypertension (n = 869), of subjects with high fibrinogen plasma levels (>3.53 g x l(-1), mean value) and hypertension (n = 599) and of former and present smokers with high fibrinogen and hypertension (n = 452). An association of the gene variation with MI was not detected. CONCLUSIONS: The present data indicate that the 4G/4G genotype of the PAI-I gene polymorphism is an independent risk factor for coronary artery disease and that the additional presence of major cardiovascular risk factors accelerates the risk for this disease.     

Tissue plasminogen activator protects hippocampal neurons from oxygen-glucose deprivation injury

We have previously shown that tissue plasminogen activator (tPA) participates in the neurotoxicity of microglial conditioned medium (MgCM). Killing of hippocampal neurons by MgCM was prevented by both plasminogen activator inhibitor-1 (PAI-1) and anti-tPA antibody. An N-methyl-D-aspartate (NMDA) receptor blocker protected neurons from MgCM, suggesting that this subtype of glutamate receptor is involved. Whereas glutamate receptor-mediated events are important in cerebral ischemia and tPA has previously been shown to enhance excitotoxicity in hippocampus, we hypothesized that tPA would exaggerate oxygen glucose deprivation (OGD) injury in cultures of hippocampal neurons. Dissociated rat hippocampal cells were grown under conditions designed to optimize neuronal growth while minimizing glial replication. At 7--10 days, cultures were subjected to OGD for 2.5 hr. Recombinant human tPA (1,000 IU) was added immediately after OGD. Viability was assessed 24 hr later. Viable, apoptotic, and necrotic cells were classified and quantified based on staining patterns of acridine orange and ethidium bromide under fluorescence microscopy. tPA alone did not alter neuronal integrity. OGD produced significant neuronal death (viability reduced by 45%, P < 0.001). tPA completely protected OGD-exposed cultures. Potential mechanisms of tPA protection were explored. Whereas tPA antibody abolished the protective effect of tPA, its proteolytic inhibitor PAI-1 did not alter the effect. The effect of tPA was tested in separate free radical and excitatory amino acid insults. It did not protect neurons from hydrogen peroxide (1 microM), S-nitro-acetylpenicillamine (10 microM), glutamate (50 microM), or NMDA (10 microM) damage but significantly attenuated injury caused by 250 microM kainate. We conclude that tPA is capable of protecting hippocampal neurons from OGD by a nonproteolytic action. The mechanism of protection was not defined, although attenuation of AMPA/kainate glutamate receptors may play a role.     

Immunoaffinity purification of tissue plasminogen activator from serum-supplemented conditioned media using monoclonal antibody

Tissue plasminogen activator (tPA) was purified from serum-supplemented culture media in a single-step immunoaffinity chromatographic procedure. This was done by producing a monoclonal antibody to tissue plasminogen activator. The antibody is a murine IgG1, kappa light chain molecule. The antibody was coupled to CNBr-activated Sepharose 4B. Tissue plasminogen activator was isolated from serum supplemented tissue culture media by immunoaffinity chromatography using a relatively high ionic strength equilibration buffer and an acidic elution buffer.