Tissue plasminogen activator: Comparison of dose and route of administration in a rabbit model of thromboembolic stroke

Abstract

The thrombolytic activity of tissue plasminogen activator was evaluated in a rabbit model of thromboembolic stroke using both various concentrations (3, 5, and 10 mg/kg; 20% bolus with the remaining 80% given over 30 min.) and routes of administration (intravenous versus regional intra-arterial). An autologous tin-tagged clot was embolized to the brain via the carotid artery. Tissue plasminogen activator was then given at the doses and routes noted (n = 3 in all groups). Thrombolytic activity was followed by serial x-rays of the tin-laden clot over a four-hour period. The brains were then removed and subjected to gross inspection. Only the intravenous dose of 5 mg/kg tissue plasminogen activator produced greater than 50% clot lysis in all animals. Doses of t-PA higher (10 mg/kg) or lower (3 mg/kg) than this were less effective in producing thrombolysis, demonstrating greater than 50% clot lysis in only one animal of each group. We conclude that in this model of thromboembolic stroke the intravenous administration of tissue plasminogen activator is more effective than intra-arterialand that the optimal dose is in the range of 5 mg/kg. [Neurol Res 1993; 15: 405-408]     

Tissue plasminogen activator-mediated thrombolysis of cerebral emboli and its effect on hemorrhagic infarction in rabbits

Tissue plasminogen activator (tPA) dissolves intravascular thrombus and restores blood flow after thromboembolic vascular occlusion. The utility of this agent for treatment of stroke in humans may be limited by post-reperfusion hemorrhagic complications. We studied tPA-mediated thrombolysis in an animal model of cerebrovascular occlusion in order to determine what factors, if any, predispose tPA-treated animals to suffer hemorrhage. Small blood clot emboli were injected into the internal carotid arteries of rabbits. Angiograms confirmed occlusion of the middle cerebral artery or internal carotid artery in 100% of subjects. tPA or saline was administered as a 30-minute infusion at various times after embolization. Hemorrhage rates were similar in all groups regardless of treatment. tPA increased the prothrombin time and the thrombin time but not the partial thromboplastin time. There was no correlation between these changes in blood coagulation and the finding of cerebral hemorrhage. We observed a significant association between stroke severity and cerebral hemorrhage. We conclude that tPA treatment successfully causes thrombolysis of cerebral emboli without causing an increase in the incidence of cerebral hemorrhage in rabbits.     

Thrombus age and tissue plasminogen activator mediated thrombolysis in rats

Thrombolytic efficacy is directly related to thrombus age. We used recombinant tissue plasminogen activator (rt-PA), Streptokinase (SK) and Urokinase (UK) on a seven days old inferior vena cava thrombus model. "In vitro" clot lysis assays with fibrinogen-I125 were also evaluated with the same agents at 1, 3 and 7 days. Fibrinogen, D-D dimer and t-PA were measured. Experiments with 40 controls and 27 rt-PA treated animals showed a significant decrease in thrombus weight (8.5 +/- 1.1 mg) vs. (4.2 +/- 0.6 mg) (p less than 0.01). Fibrinogen concentration in rt-PA group decreased significantly (1032 +/- 123 mg/dl) vs. (202 +/- 32 mg/dl) (p less than 0.001). "In vitro" rt-PA showed a marked lytic effect in a wide range (100-4 IU/ml). Fibrin selective agents as rt-PA may be more effective than non selective ones in the treatment of fully developed thrombus.     

Tissue plasminogen activator expression in meningiomas and glioblastomas

Objectives:Enzyme-linked immunosorbent assay (ELISA) and Western blotting techniques were used to investigate and compare the expression of tissue plasminogen activator (tPA) in benign (meningioma) and malignant (glioblastoma) human brain tumours.Methods:A total of 22 tumour samples comprising 11 meningiomas and 11 glioblastomas with adjacent peritumoural tissue were analysed.Results:The mean tPA content of meningiomas was approximately half that of glioblastomas (55.40 (S.D. 34.58) versus 106.98 (S.D. 43.82) ng/ml, p = 0.006). Comparing tPA quantity in tumour and peritumoural tissue, there was a significant difference for meningiomas (55.40 (S.D. 34.58) versus 28.35 (S.D. 22.55) ng/ml, p = 0.05), but no difference for glioblastomas (106.98 (S.D. 43.82) versus 84.23 (S.D. 57.39) ng/ml, p = 0.32). Comparing tumour with normal brain tissue, there was no difference for meningiomas (55.40 (S.D. 34.58) versus 33.08 (S.D. 21.55) ng/ml, p = 0.22), but a significant difference for glioblastomas (106.98 (S.D. 43.82) versus 33.08 (S.D. 21.55) ng/ml, p = 0.004).Western blotting showed that in the meningioma group, the molecular weight pattern was constant with a dominant well-defined band at 41 kD. Peritumoural tissue demonstrated two bands, with the stronger band at 41 kD and a slightly weaker band at 71 kD.In the glioblastoma group, there was more heterogeneity, with a dominant 41 kD band found in all tumour and peritumoural samples, together with additional bands at 34, 58 and 66 kD.Conclusion:These results indicate that (1) tPA is present in larger quantities in glioblastoma compared to meningioma and normal brain, (2) tPA quantity is not significantly different in the peritumoural tissue adjacent to glioblastoma but is significantly less for meningioma, and (3) tPA is expressed in more heterogenous forms in glioblastoma. This present study therefore suggests that the expression of tPA in a brain tumour may be an additional prognostic factor in terms of its malignant and invasive potential.     

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.     

Tissue plasminogen activator in nervous system function and dysfunction

The extracellular protease tissue plasminogen activator (tPA) has been implicated in various normal and pathological situations in the mammalian nervous system. The availability of (i) transgenic and knock-out mice in which the expression level of tPA can be widely varied, (ii) in vivo models for studying function and disease, and (iii) culture models for examining cell behavior, has allowed a detailed evaluation of many of these proposed functions. This chapter summarizes the current state of knowledge of possible roles for the tPA/plasminogen system in neuronal function and dysfunction.     

The endotoxin-induced plasminogen activator inhibitor-1 increase in rabbits is not tumor necrosis factor-alpha dependent and can occur in the absence of interleukin-1beta

The plasminogen activator inhibitor-1 (PAI-1)-dependent fibrinolytic inhibition occurring in endotoxemia contributes to disseminated intravascular coagulation (DIC). Previous findings suggest that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) are responsible for the increase in the level of PAI-1. These observations usually arose from mild endotoxemia models. We analyzed the effect of FR167653, an inhibitor of the TNF-alpha/IL-1beta production, on the PAI-1 levels in rabbits given endotoxin at a dose sufficient to induce DIC: the steep plasma PAI-1 increase was not attenuated by FR167653, in spite of achieving efficient inhibition of the TNF-alpha production. No IL-1beta was detected during endotoxemia. These results suggest that PAI-1 increase might be independent of TNF-alpha and IL-1beta. If these findings applied to humans, therapeutic intervention directing these cytokines would not be useful for the treatment of fibrinolysis in patients with severe sepsis.     

Turnover of tissue plasminogen activator in normal and hepatectomized rabbits

The distribution, and clearance of the tissue plasminogen activator (tPA) was studied in rabbits, rats and mice. Following an intravenous injection of I-labelled tPA a large portion of the radioactive dose was rapidly accumulated in the liver. After one hour the radioactivity in the liver was less than 5 per cent of the injected dose. The highest activity was then found in the intestine, stomach and in the blood. Gel filtration of plasma taken one hour after the injection revealed that the major part (60%) of the radioactivity was present as low molecular weight metabolites or free iodine. The remaining activity was present as high molecular weight inhibitor complexes (26%) or as free tPA (15%). In order to study in vivo reactions between tPA and plasma inhibitors without hepatic interference, plasma turnover was also studied in hepatectomized rabbits. High amounts of radioactivity remained in the plasma after one hour. Gel filtration of this plasma revealed that 54 per cent of the radioactivity was bound to inhibitors, 34 per cent circulated as free tPA while a minor portion (12%) was found as free iodine or metabolites. The half-life of fibrinolytically active tPA in hepatectomized rabbits was 40 minutes compared to 2 minutes in intact rabbits. The increase in the half-life of tPA in hepatectomized rabbits also resulted in an improved thrombolytic effect after treatment with 0.5 mg tPA.     

Elimination of native and carbohydrate-modified tissue plasminogen activator in rabbits

The elimination of native and carbohydrate-modified tissue type plasminogen activator (t-PA) after an i.v. bolus injection was studied in rabbits. t-PA with a low content of mannose (man-t-PA) was obtained by treatment with alpha-mannosidase, which cleaves terminal mannose from the carbohydrate side chains of the molecule. About 50% of the total mannose was removed from the native t-PA. Clearance of t-PA in rabbits was followed by measurements of both the fibrinolytic activity of the activators and the radioactivity of the iodine-labelled compounds. A biphasic mode of elimination of the fibrinolytic activity as well as the radioactivity was found with both the native and the carbohydrate modified t-PA. The initial half-life (t 1/2 alpha) was the same, about 1.5 minutes, for both compounds, irrespective of method of analysis. The late half-life (t 1/2 beta) was also the same, about 15 minutes, for both compounds. However, the beta-elimination of native t-PA could only be determined accurately from radioactivity data in the dosages tested, i.e. up to 2 mg of t-PA. No dose dependency in the elimination pattern was observed. In gel filtration experiments, with plasma from the rabbits, it was demonstrated that in addition to fibrinolytically active t-PA and man-t-PA, both fibrinolytically inactive high molecular weight complexes and low molecular weight degradation products of the activators were present in plasma after injection of the compounds. The second phase of elimination (beta) was much more pronounced after mannosidase treatment of the t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue plasminogen activator gene expression in multiple sclerosis brain tissue.

Recent studies have implicated tissue-type plasminogen activator (tPA) in neurodegeneration. We studied multiple sclerosis (MS) brain tissue for tPA gene and protein expression in comparison with reference tissue, by in situ hybridisation and immunohistochemistry. MS is characterised by demyelination in the central nervous system. In this study, neuronal cell bodies in MS brain showed high expression of tPA mRNA and protein, while in reference brains, staining for protein and mRNA expression were very low in neurons and mostly restricted to blood vessel walls. In MS, there was an additional staining of mononuclear cells within perivascular cuffs and foamy macrophages within demyelinating plaques. In view of evidence that the final process of demyelination in MS is thought to be enzyme-mediated, our work suggests the involvement of tPA and by inference plasmin, in the demyelinating process. Blocking tPA or plasmin activity may be a potentially beneficial therapeutic approach in MS.     

Tissue plasminogen activator for acute stroke in everyday clinical practice.

Thrombolysis for acute ischemic stroke has become a reality. The aim of our study was to assess the opportunity and practicality of establishing acute stroke treatment in a hospital that did not participate in acute stroke treatment trials, as well as to prospectively analyze 2 groups of patients who reached the Emergency Department (ED) within 3 hours who were either treated or not treated with tissue plasminogen activator (t-PA). The average score for severity of neurological deficits for the patients who received t-PA was 14 on the National Institute of Health Stroke Scale (NIHSS). We compare this group with 18 patients who did not receive t-PA but had similar NIHSS scores (13.9 average). Both groups were matched for age and other comorbidity factors. We concluded that the establishment of an acute stroke treatment algorithm is possible de novo in a hospital that is equipped with computed tomography (CT) and neurosurgery services. The number of patients who can receive t-PA treatment is limited by the strict inclusion and exclusion criteria. Prolonged door-to-needle time was caused by delays in CT interpretation, processing of laboratory results, and stabilization of blood pressure. Patients who received t-PA had a shorter length of stay, were more independent, and had a better survival rate after 1 year. Our findings were in agreement with the National Institute of Neurological Disorders and Stroke (NINDS) Stroke Study that led to the approval of the use of t-PA in the treatment of acute ischemic stroke.     

Tissue plasminogen activator is endocytosed by mannose and galactose receptors of rat liver cells

Experiments were carried out to characterize the specificity of uptake of tPA in rat liver cells. Endocytosis in liver endothelial cells of the native carbohydrate variants of tissue plasminogen activator (tPA), and tPA inactivated by diisopropyl fluorophosphate was found to be competitive, suggesting that the determinant being recognized by these cells is different from the active site. Fibronectin and urokinase, which show partial homology with tPA, did not compete with tPA for uptake in liver endothelial cells. Hyaluronic acid, collagen, or IgG, which are endocytosed by specific receptors in liver endothelial cells, did not interfere with the uptake. Reduced endocytosis by liver endothelial cells was observed with tPA modified in the carbohydrate side chains, suggesting that these structures are important for uptake. Ovalbumin, mannan, mannose, fructose, and EDTA, but not galactose, effectively inhibited uptake in liver endothelial cells of both native and diisopropyl fluorophosphate-inhibited tPA, but had very little effect on the uptake of tPA modified in the carbohydrate side chains. Endocytosis of native tPA by parenchymal cells could be inhibited by galactose, ovalbumin, and EDTA, but not by mannose. These results suggest that endocytosis of tPA by liver endothelial cells and parenchymal cells is mediated by the mannose and galactose receptors, respectively.     

Function of tissue-type plasminogen activator releaser on vascular endothelial cells and thrombolysis in vivo

The effect of monosodium[2-(6-hydroxynaphthalen-2-yl)-6-methylpyrimidin-4-yloxy]acetate dihydrate (JTV-926) on fibrinolysis was investigated in vitro and in vivo. JTV-926 released tissue-type plasminogen activator (t-PA) from human vascular endothelial cells in a dose-dependent manner. The thrombolytic effect of JTV-926 was studied using three animal thrombosis models; a photo-irradiation-induced mouse carotid artery thrombosis model, a photo-irradiation-induced rat femoral artery thrombosis model and a thrombin-induced rat venous thrombosis model. In the mouse thrombosis model, t-PA deficient mice (t-PA-/- mice) and their wild-type (t-PA+/+) were used. JTV-926 was injected as a bolus 30 min after the interruption of blood flow by an occlusion thrombi. Blood flow was continuously monitored for 180 min after intravenous administration of JTV-926 (1 mg/kg). Although the recanalization rate of the occluded artery was 37.5% in t-PA +/+ mice with the vehicle control, it increased to 75% in t-PA+/+ mice after JTV-926 administration. However, when JTV-926 was administrated in t-PA-/- mice, vascular recanalization was not observed in any arteries. In the photo-irradiation-induced rat femoral artery thrombosis model, intra-duodenal administration of JTV-926 induced thrombolysis. Moreover, in the thrombin-induced rat venous thrombosis model, the dose-dependent thrombolysis was also observed by oral administration of JTV-926. It was suggested that JTV-926 revealed a sufficient thrombolytic effect through the absorption from the intestine. Thus, a newly synthesized compound, JTV-926 induced t-PA release from vascular endothelial cells and effective thrombolysis in vivo.     

The effect of thrombin inhibitors on tissue plasminogen activator induced thrombolysis in a rat model.

Successful coronary thrombolysis depends on rapidly restoring blood flow and maintaining patency of the infarct-related artery. Although widely used as an adjunct to lytic therapy, heparin is limited in its ability to produce these effects. Since the limitations of heparin may reflect its inability to inactivate clot-bound thrombin, we developed a rat model of tissue plasminogen activator (t-PA) induced thrombolysis to compare doses of heparin, hirudin, hirulog (a synthetic hirudin-derived peptide), and D-Phe-Pro-ArgCH2Cl (PPACK) that produced a 4-fold prolongation of the baseline activated partial thromboplastin time (APTT) with saline in terms of their ability to accelerate thrombolysis and to prevent reocclusion. A thrombus rich in red cells and fibrin was formed in the distal aorta by applying an external constrictor after denuding the endothelium with a balloon catheter. Thrombolysis was induced with t-PA (1 mg/kg bolus, followed by 1 mg kg-1 h-1 over 30 min) and the rats were then randomized to receive a concomitant 80 min infusion of a thrombin inhibitor or saline. By continuously monitoring blood flow and pre- and post-stenotic blood pressures, the time to clot lysis, and the number of reocclusions were determined. Compared to saline, heparin had no significant effect on these variables.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of synergism between tissue-type plasminogen activator and urokinase on plasminogen activation rate in plasma

Effects of tissue-type plasminogen activator (t-PA), urokinase(u-PA) and their combinations on plasminogen activation rate (PAR) in plasma, were investigated. T-PA and u-PA over concentrations range of 10 U/ml to 50 U/ml induced a linear, concentration dependent increase in PAR. Combinations of t-PA and u-PA in ratios of 3/1,1/1 and 1/3 induced additive but not synergistic effect in the activation of plasminogen. We conclude, therefore, that t-PA and u-PA do not act synergistically in the activation of plasminogen in plasma .     

Tissue plasminogen activator in central nervous system physiology and pathology

Although conventionally associated with fibrin clot degradation, recent work has uncovered new functions for the tissue plasminogen activator (tPA)/plasminogen cascade in central nervous system physiology and pathology. This extracellular proteolytic cascade has been shown to have roles in learning and memory, stress, neuronal degeneration, addiction and Alzheimer's disease. The current review considers the different ways tPA functions in the brain.     

Tissue plasminogen activator and plasminogen activator inhibitor in patients with acute ischemic stroke: relation to stroke etiology

Recent studies suggest that high plasma levels of tissue-type plasminogen activator (tPA) and its inhibitor (plasminogen activator inhibitor-1, PAI-1) are markers of an increased risk of atherothrombotic ischemic events such as stroke and myocardial infarction. In this prospective study, we measured tPA antigen, PAI-1 antigen and activity, as well as tPA/PAI-1 complex in patients with acute stroke. Stroke subtypes were classified according to the TOAST criteria. From 132 consecutively screened patients, 89 (100%) were enrolled in this study, including 42 patients (47%) with large artery atherosclerosis (LAA), 32 (36%) with small vessel occlusion (SVO), and 15 (17%) with cardioembolism (CE). Nineteen age-matched neurologic patients without manifestations of cerebrovascular disease served as control subjects (CS). Patients with acute stroke had significantly higher plasma levels of tPA antigen (p < 0.001), PAI-1 antigen (p < 0.05) and PAI activity (p < 0.05) than patients in the control group. t-PA antigen, PAI activity and tPA/PAI-1 complex levels were similar regardless of stroke etiology. Only PAI-1 antigen was lower in patients with cardioembolic stroke than in stroke patients with LAA (p < 0.05). Plasma tPA antigen, PAI-1 antigen, and PAI activity are significantly increased in patients with acute ischemic stroke. Except for PAI-1 antigen, this increase appears not to be related to the underlying stroke etiology.     

Clearance of a novel recombinant tissue plasminogen activator in rabbits

The pharmacokinetic properties of hPA(B), characterized by the insertion of a urokinase kringle coding region before the double kringle of tPA plus the complete tPA coding region, were investigated and compared to those of melanoma tPA (mtPA). Mean peak plasma concentrations at the end of infusion were 4.7 micrograms/ml for hPA(B) and 4.6 micrograms/ml for mtPA. The pharmacokinetics of both hPA(B) and mtPA showed a biexponential disappearance from plasma which is consistent with a two-compartment model of t 1/2 (lambda 1) = 2 minutes, t 1/2 (lambda 2) = 58 minutes for hPA(B), and t 1/2 (lambda 1) = 2.2 minutes, t 1/2 (lambda 2) = 61 minutes for mtPA. However, this very fast decaying lambda 1 phase of mtPA lasted five times longer than that of hPA(B) which resulted in very low concentrations of mtPA. Thus, hPA(B) exhibited larger AUC, slower clearance rate, and smaller volume of distribution (P less than 0.01) than those of mtPA. The fibrinolytic activity of hPA(B) in rabbit plasma as determined by zymography lasted up to 120 minutes after the end of infusion as compared to that of 2 minutes for mtPA. This indicates that mtPA, despite its t 1/2 (lambda 2) being similar to that of hPA(B), is no longer at physiologically meaningful concentrations at the start of the lambda 2 phase.     

Tissue plasminogen activator administration does not result in clinically relevant neuropsychological dysfunction in patients with myocardial ischemia

BACKGROUND: Recent evidence from animal models has demonstrated that tPA can be neurotoxic to the hippocampus and cerebellum. Since tPA is used in patients with myocardial infarction and has been approved for the treatment of acute ischemic stroke, we carried out a pilot study to investigate whether its administration could result in any clinically relevant neuropsychological dysfunction, with particular attention to those structures most involved in the animal model. PATIENT AND METHODS: Patients with acute myocardial infarction (AMI) who were subjected to thrombolysis with r-tPA and without evidence of prior or concomitant neurological-neuropsychological damage, were eligible. Controls consisted of patients with AMI not candidate for thrombolysis or patients with severe angina admitted to the intensive care unit. A detailed neurological and neuropsychological evaluation was performed, which included mood, visual and verbal memory, psychomotor speed processing and motor dexterity and visuomotor sequence learning. RESULTS: Ten patients and 13 controls were included. No significant differences between both groups were found, although all the subjects performed in the lower limits of the normal range on the Purdue pegboard test (motor dexterity) and digit-symbol test (psychomotor speed). There was no evidence of depression, as assessed by the Hamilton scale, that could have interfered with performance. Declarative memory (Rey auditory-verbal learning test) as well as visual memory (Rey-Osterrieth figure copy) and visuomotor sequence learning (serial reaction time task) were normal, without between-group differences. CONCLUSIONS: tPA administration at the usual doses did not result in detectable neuropsychological abnormalities in patients with myocardial ischemia.