doi: 10.1161/01.STR.0000258100.04923.84
(Stroke. 2007;38:1036.)
© 2007 American Heart Association, Inc.
Original Contributions |
Recombinant Desmodus rotundus Salivary Plasminogen Activator Crosses the Blood–Brain Barrier Through a Low-Density Lipoprotein Receptor-Related Protein-Dependent Mechanism Without Exerting Neurotoxic Effects
From INSERM (J.P.L.-A., B.D.R., C.A., E.M., C.O., D.V.), INSERM-Avenir, Caen Cedex, France, Université de Caen Basse-Normandie, Caen Cedex, France, and GIP Cyceron, Caen, France; PAION Deutschland GmbH (K.-U.P.), Aachen, Germany; Laboratoire de Physiopathologie de la Barrière Hémato-Encéphalique (V.B., R.C.), Université d’Artois, Faculté des sciences Jean Perrin, France.
Correspondence to Denis Vivien or José P. López-Atalaya, INSERM-Avenir, GIP Cyceron, Bd H.Becquerel, BP 5229, 14074 Caen, France. E-mail vivien{at}cyceron.fr or jose.lopez@umh.es
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Abstract |
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Background and Purpose— Desmoteplase, a recombinant form of the plasminogen activator DSPA
1 from Desmodus rotundus, may offer improved clinical benefits for acute ischemic stroke treatment over the current therapy, recombinant tissue plasminogen activator (rtPA). Accumulating evidence suggests that clinical use of rtPA could be limited by unfavorable properties, including its ability to cross the blood–brain barrier (BBB), thus potentially adding to the pro-excitotoxic effect of endogenous tPA in cerebral parenchyma. Here, to investigate whether desmoteplase may display a safer profile than the structurally-related tPA, both agents were compared for their ability to cross the BBB and promote neurotoxicity. Methods— First, the passage of vascular DSPA and rtPA was investigated in vitro in a model of BBB, subjected or not to oxygen and glucose deprivation. Second, we studied DSPA- and rtPA-mediated effects in an in vivo paradigm of excitotoxic necrosis.
Results— The rtPA and desmoteplase cross the intact BBB by LRP-mediated transcytosis. Under conditions of oxygen and glucose deprivation, translocation rates of both compounds increased; however, unlike rtPA, desmoteplase transport remained LRP-dependent. Additionally, neither intracerebral nor intravenous desmoteplase administration enhanced NMDA-induced excitotoxic striatal damage in vivo. Interestingly, intravenous but not intrastriatal coadministration of desmoteplase and rtPA reduced the pro-excitotoxic effect of rtPA.
Conclusions— We show that desmoteplase crosses the BBB but does not promote neuronal death. Moreover, intravenous administration of desmoteplase antagonizes the neurotoxicity induced by vascular rtPA. This action may be caused by competition of desmoteplase with rtPA for LRP binding at the BBB, thus effectively blocking rtPA access to the brain parenchyma.
Key Words: blood–brain barrier • desmoteplase • excitotoxicity • stroke thrombolysis
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Introduction |
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Recombinant tissue-type plasminogen activator (rtPA) is a thrombolytic agent currently used in clinical practice for the treatment of peripheral and cerebral ischemia; however, the overall clinical benefit of rtPA, especially in acute ischemic stroke, has been limited by the risks associated with a delay in drug administration. Beyond the critical period of three hours post-symptom onset,1 the risk for symptomatic intracerebral hemorrhage outweighs the benefit of reperfusion with rtPA.2
These clinical limitations have inspired a search for alternative plasminogen activators (PAs). Desmoteplase is a recombinant PA derived from DSPA1, a highly fibrin-specific PA, present in the saliva of the vampire bat, Desmodus rotundus3. Preclinical evidence suggests that desmoteplase may offer clinical benefits over rtPA, including a reduced risk for symptomatic intracerebral hemorrhage, improved safety, and a longer treatment window. In two recent phase II clinical stroke trials, patients were administered desmoteplase within 3 to 9 hours of acute ischemic stroke symptom onset. Results suggest higher rates of reperfusion and improved clinical outcomes at 90 days compared with placebo, with a very low rate of symptomatic intracerebral hemorrhage.4,5 The DIAS-2 phase III clinical trial is currently underway to investigate the efficacy of desmoteplase.
One of the factors likely to contribute to the favorable clinical result in the Phase II trials is the low or absent neurotoxic potential of desmoteplase. In mice, when coinjected with NMDA into the striatum6 or when administered intravenously 24 hours after NMDA-induced injury,7 desmoteplase, in contrast to rtPA, does not promote excitotoxin-induced neuronal death. This may be of particular importance given that endogenous tPA released within the cerebral parenchyma secondary to ischemia could have detrimental effects including enhancement of neurotoxic processes.8,9 Furthermore, while effective reperfusion by vascular rtPA can be achieved, the use of this serine protease has been associated with blood-brain barrier (BBB) leakage in animal stroke models.10 Recently it has been demonstrated that rtPA injected intravenously can reach the brain parenchyma by crossing the intact BBB.11 This effect is independent from its proteolytic activity and mediated by a receptor-dependent mechanism, identified as a member of the LRP receptor family.11 Moreover, the passage of rtPA across the BBB is dramatically increased and no longer LRP-dependent under conditions that mimic ischemia (oxygen and glucose deprivation).12
Here, in vitro and in vivo experiments have been performed to evaluate the ability of both rtPA and desmoteplase a) to cross the BBB and b) to enhance, potentially, NMDA excitotoxicity.
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Materials and Methods |
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Reagents
Human recombinant tPA (Actilyse) was from Boehringer Ingelheim (France), NMDA from Tocris (U.K.). RAP was provided by Dr. Bu. Desmoteplase was provided by PAION (Germany).
Animals
Male Sprague Dawley rats (270 to 330 g) were housed in a temperature-controlled room on a 12-hour light/12-hour dark cycle, with food and water ad libitum. Experiments were performed in accordance with French (act no. 87 to 848; Ministère de l’Agriculture et de la Forêt) and European Communities Council (Directives of November 24, 1986, 86/609/EEC) guidelines for the care and use of laboratory animals.
Striatal Excitotoxic Lesions
Rats were anesthetized with isoflurane (5%, maintenance 2% in oxygen/N2O (1:3) at 0.8 l/min). Body temperature was maintained at 37±0.5°C. Injection pipette (internal diameter 0.32 mm and calibrated at 15 mm/µL; Hecht Assistent, Germany) was stereotaxically implanted in the right striatum (3.5 lateral and 5.5 ventral to the bregma). NMDA (50 nmol) was injected in a volume of 1 µL and the pipette was removed 3 minutes later. In the first set of experiments, excitotoxic treatment was complemented after 15 minutes by intravenous injection of rtPA (1 mg/kg), desmoteplase (1 mg/kg), rtPA+desmoteplase (1 mg/kg each), tPA vehicle (L-Arg 35 mg/kg, phosphoric acid 10 mg/kg and polysorbate 80 0.2%) or desmoteplase vehicle (glycine 4 mg/kg and mannitol 10.64 mg/kg). In the second set of experiments, NMDA was coinjected into the striatum with rtPA (3 µg), desmoteplase (3 µg), both (3 µg each) or the corresponding vehicles, all in a volume of 1 µL.
Histological Analysis
After 24 hours, rats were euthanized and the whole brain was removed and frozen in isopentane. For volume analysis, one coronal section (20 µm) out of every 20 was stained with thionine and analyzed, covering the entire lesion. Regions of interest were determined using a stereotaxic atlas for the rat (Paxinos and Watson), and an image-analysis system (BIOCOM RAG 200, Paris, France) was used to measure the lesion given by the nonstained area.
BBB Experiments
Transport across the BBB was studied using a previously characterized in vitro model (Figure 1A), consisting of a coculture of endothelial and glial cells and shown to closely mimic the in vivo BBB.13 Briefly, bovine brain endothelial cells were isolated from brain capillaries, grown in DMEM supplemented with 10% heat-inactivated calf serum and 10% horse serum (Hyclone Laboratories, USA), 2 mmol/L glutamine, 50 µg/mL gentamicin and basic fibroblast growth factor (1 ng/mL, added every other day). Primary glial cultures were isolated from newborn rat cerebral cortex. After removing the meninges, the brain tissue was gently forced through a nylon sieve. DMEM supplemented with 10% fetal calf serum, 2 mmol/L glutamine, and 50 µg/mL of gentamycin was used for the dissociation of cerebral tissue and development of glial cells. Three weeks after, glial cultures were confluent and composed of astrocytes (
60%), oligodendrocytes and microglial cells. Coated filters were then placed in six-well dishes containing glial cells for coculture. Endothelial cells were plated on the upper side of the filter at a concentration of 4·105 cells/mL. The coculture medium was the same as that for brain capillary endothelial cells. Experiments were performed 5 days after confluence. rtPA (0.31 µmol/L; 20 µg/mL) and/or desmoteplase (0.38 µmol/L; 20 µg/mL) were added to the upper side of endothelial cells (luminal compartment). Abluminal (lower side) and luminal media were harvested after the 2 hour transport experiments. The concentration of 0.3 µmol/L of rtPA and DSPA was chosen based on our previous studies.11,12
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The passage of the paracellular marker [14C]-sucrose was determined in parallel to control for possible effects of the compounds on the coherence of the endothelial monolayer.
Oxygen and Glucose Deprivation Experiments
Oxygen and glucose deprivation (OGD) experiments were performed using the in vitro BBB model described previously.14 For OGD experiments, cells were placed for 4 hours in a 37°C oven at 0% O2, 5% CO2, 95% N2 using Gas Pack Pouch bags (Becton Dickinson); for normoxic controls, the medium was equilibrated with air and contained 1 g/L glucose. After preincubation, transport of rtPA and desmoteplase was studied keeping control batches in a normoxic environment and OGD batches under an airtight glove box filled with nitrogen at 37°C (Forma Scientific). In all experiments the pH of the medium was unchanged during normoxic or OGD conditions. Upper and lower endothelium side media were harvested after 2 hours.
Plasminogen-Casein Zymography
Zymography was performed by adding plasminogen (4.5 µg/mL) and casein (1%) to a 15% SDS polyacrylamide gel. Electrophoresis was performed at 4°C. Gels were washed with Triton X-100 (2.5%) and incubated at 37°C. Incubation time for rtPA zymography was 1 to 2 hour, whereas 8 to 9 hours were required for desmoteplase, reflecting the extremely low intrinsic proteolytic activity of desmoteplase in the absence of fibrin.15 Caseinolytic bands were visualized after Coomassie staining.
Fluorometric Assay
Fluorogenic substrate (Spectrozyme XF444, American Diagnostica, USA) at 5 µmol/L was incubated with 50 µL of medium in a final volume of 100 µL. Measurements were performed at 37°C over 2 hours using a multiplate reader (Chameleon, Hidex, Finland).
Statistical Analysis
Data were expressed as mean±SD, and statistical analyses consisted in Student t test for comparisons between two groups and an ANOVA followed by Bonferroni post hoc test for comparisons among multiple groups.
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Results |
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BBB Permeation of rtPA and Desmoteplase Does Not Result from Unspecific BBB Damage
BBB permeation was assessed by performing zymography and fluorogenic assays. Confirming previous observations,11,12 both assays showed a significant transendothelial translocation of active rtPA after 2 hours. Interestingly, desmoteplase activity was detected in the lower side of endothelium compartment after that period (Figure 1A and 1B). Both PAs were permeant at similar rates, but these rates were substantially slower than those observed with cell-free filters, indicating restriction of permeation by the endothelial cell layer (Figure 1B). Under control conditions, the BBB was practically impermeable to sucrose. Neither rtPA nor desmoteplase influenced the basal passage of sucrose, suggesting that permeation of the two drugs was not due to nonspecific BBB damage (Figure 1C).
OGD Increases the Passage of Both rtPA and Desmoteplase Across the BBB in vitro
The passage of both PAs under control conditions and in a setting designed to mimic ischemia (ie, after 4 hours of OGD) was compared. Zymography (Figure 2A and 2C) and corresponding densitometric analyses (Figure 2B and 2D) showed that the passage of both thrombolytic agents was significantly exacerbated by OGD. Similar results were obtained with the fluorogenic assay (Figure 2E and 2F). Sucrose permeability, shown in the same plot, also was increased to similar extents.
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RAP Antagonizes the Ability of Both rtPA and Desmoteplase to Cross the Intact BBB
As previously shown, rtPA crosses the intact BBB by LRP-mediated transcytosis.11 The LRP antagonist RAP (500 nM) reduced the passage through endothelium of both rtPA (Figure 3A and 3B) and desmoteplase (Figure 3C and 3D) by 50%. RAP had no effect on the permeability of sucrose (0.68±0.06 versus 0.69±0.11 x10–3 cm/min). Together, these data indicate that desmoteplase, like rtPA, could reach the brain parenchyma in the absence of BBB leakage. Furthermore, rtPA and desmoteplase likely share the same mechanism to cross the intact BBB since their coapplication to the upper-side of endothelial cells reduced the amount of bio-active rtPA crossing the BBB (Figure 3E and 3F).
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Similar experiments were conducted under ischemic-like conditions. As previously demonstrated,12 RAP did not inhibit the passage through endothelium of rtPA in the setting of OGD (Figure 4A and 4B). By contrast, RAP significantly decreased passage of desmoteplase (Figure 4C and 4D), suggesting an LRP-dependent translocation under ischemic conditions.
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Desmoteplase Does Not Exacerbate NMDA-induced Neuronal Death
The effects of intravenous injection of rtPA and desmoteplase were compared in a rat model of excitoxicity induced by injection of NMDA into the striatum (Figure 5). In the absence of brain injury, intravenous injection of rtPA or desmoteplase failed to produce striatal damage. Further, neither rtPA nor desmoteplase vehicles injected intravenously altered the extent of NMDA-induced striatal injury, ruling out any nonspecific effect.
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As previously demonstrated,11 the systemic injection of rtPA (1 mg/kg) increased the striatal lesion volume seen with NMDA by almost 40% (from 32 to 42 mm3) (Figure 5). In contrast, intravenous injection of desmoteplase (1 mg/kg) failed to exacerbate NMDA-induced injury. Interestingly, intravenous coadministration of desmoteplase with rtPA, both at 1 mg/kg, suppressed the ability of rtPA to enhance NMDA-induced lesion. On a molar basis, the applied dose of desmoteplase (19.2 nmol/kg) exceeded that of rtPA (14.5 nmol/kg). No microhemorrhages were detected in animals treated with rtPA and/or desmoteplase.
To confirm the absence of neurotoxic effects of desmoteplase, we coinjected NMDA with one of the thrombolytic agents. rtPA increased NMDA-induced neuronal death in the striatum, whereas the same dose of desmoteplase did not. In addition, we found that the coapplication of desmoteplase did not alter the extent of striatal injury induced by NMDA plus rtPA (Figure 6).
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Discussion |
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To date, the only approved therapy for ischemic stroke is early intravenous administration of rtPA. Despite the advantage of reperfusion therapy, clinical and experimental evidence indicate that the achievable benefit of rtPA-driven thrombolysis is limited by the narrow treatment window, as the incidence of symptomatic brain hemorrhage2 increases beyond the 3-hour time-window approved for rtPA administration. Among the numerous attempts to establish safer thrombolytic approaches, recombinant DSPA
1 (desmoteplase) stands out as a promising alternative, as suggested by the results from two recently completed successful phase II trials.4,5 Although the beneficial thrombolytic activity of rtPA has been clearly demonstrated, increasing evidence suggests that by promoting excitotoxic neurodegeneration, rtPA may have detrimental effects.8,9 These observations arising from animal models may be relevant since beneficial effects of reperfusion could be restricted to some extent. Therefore, further efforts to improve this therapeutic strategy for acute stroke may be translated into a benefit for patients.
The harmful cerebral effects of rtPA arising from animals models have suggested that tPA derived from cerebral parenchyma16 as well as tPA of vascular origin7,11,17 exert potential pro-neurotoxic effects. The fact that systemic tPA can injure the parenchyma is, at least partly, attributable to its ability to cross the blood-brain barrier.11 This finding highlights the importance of understanding interactions at the level of the neurovascular unit (endothelium, astrocyte and neuron).18
Here, we first sought to determine whether vascular desmoteplase crosses the BBB in normal and pathological conditions, and if so, by which mechanism. The passage of vascular desmoteplase was investigated in an in vitro model of BBB, either subjected to OGD or not. The in vitro BBB model used in our experimental setting consists of a coculture of endothelial cells from brain capillaries and glial cells (Figure 1A) and closely mimics the in vivo situation in features such as a high electrical resistance (up to 800 
/cm2), a very low permeability for sucrose and inulin, and constitutive tight junctions revealed by the presence of occludin, ZO-1, claudin-1, and claudin-5 immunostainings.13,14 We used a dose of 0.3 µmol/L of either rtPA or desmoteplase based on the observation that, with rtPA, such a concentration can be reached in blood during thrombolysis.19
Using this model, we have shown that under normal conditions, desmoteplase can cross the intact BBB like rtPA, and that the passage of both proteases is accelerated in a setting that mimics late ischemic conditions.
LRP is a large transmembrane scavenger receptor of the low-density lipoprotein receptor family that functions as an endocytotic receptor for a number of ligands.20 Within the central nervous system, LRP is not only expressed in neurons and astrocytes, but also at the BBB level where it mediates the transcytosis of proteins such as lactoferrin from the blood to the brain. The inhibitory effect of the LRP antagonist, RAP, shown in this report indicates that in an intact BBB, LRP mediates endothelial translocation of both rtPA and desmoteplase. Interestingly, both thrombolytic agents seem to compete for LRP in the in vitro BBB model (Figure 3E and 3F). Under OGD conditions, desmoteplase appears to cross the BBB at a rate lower than rtPA. This could be due to the fact that following OGD, rtPA can cross mainly via a nonreceptor-mediated process,12 that is not saturable, in contrast to the LRP-dependent pathway on which desmoteplase remains dependent after OGD. A possible explanation could be that desmoteplase displays a higher affinity for LRP than rtPA, a notion supported by the in vitro competition study in which equimolar concentrations of rtPA and desmoteplase were used (Figure 3E and 3F).
The principal structural difference between DSPA1 and tPA is the absence of the Kringle 2 (K2) domain.3,15 Our results suggest that K2 is not involved in the interaction of rtPA with LRP, consistent with the previous demonstration that this interaction requires the EGF domain and is independent of catalytic activity.21
Next, we aimed to characterize whether the new thrombolytic agent desmoteplase also has the harmful effects previously reported for tPA in excitotoxic models of brain injury. In this context, Chen and colleagues have investigated whether neurodegeneration after excitotoxic injury is correlated with BBB disruption. They showed that excitotoxic injury leads to a late BBB breakdown (24 hours after kainate injection) in mice, suggesting that the establishment of the lesion and the BBB breakdown are independent processes.22 Accordingly, we have recently shown that excitotoxic challenge does not alter the BBB integrity at least 2 hours after intrastriatal injection of NMDA. In these conditions, nearly all the tPA injected was cleared from circulation and found in either the brain parenchyma or in the cerebrospinal fluid. Moreover, in the same experiments, it was demonstrated that exogenous tPA can reach the brain parenchyma and influence neuronal fate in the absence of BBB breakdown.11 In the same model, we observed that in contrast to tPA, desmoteplase does not exert pro-neurotoxic effects either by iv or by intrastriatal treatment. In addition, desmoteplase appears to antagonize tPA-mediated pro-neurotoxic effects when both were coinjected by iv but not when coadministered into the striatum (Figure 5 and 6
). These findings strongly suggest a relevant in vivo competition for LRP at the BBB level between these two thrombolytic agents as demonstrated in vitro (Figure 3E).
Absence of deleterious effects associated with neurotoxicity of intravenously injected desmoteplase has already been reported in a slightly different model of striatal excitotoxic injury in mice in which the authors demonstrated that desmoteplase can gain access to the brain through a damaged BBB.7 The same group has demonstrated that intra-structure administration of desmoteplase does not restore the susceptibility of hippocampal neurons to kainate-mediated injury (and associated microglial activation) in tPA-deficient mice and does not increase NMDA-induced striatal injury.6 Our data are in agreement with their suggestion that desmoteplase is devoid of neurotoxic effects.
The same authors reported that desmoteplase, in the presence of NMDA, antagonized the ability of rtPA to enhance cellular calcium influx and death in primary cultures of cortical neurons.7 This is not easily reconciled with our present in vivo data which shows that the striatal coinjection of the two drugs did not mitigate the detrimental effect of rtPA. The reasons for this discrepancy are unknown; however, it is clear that different experimental conditions may be pertinent (eg, only a subset of neuronal cells may be sensitive to desmoteplase antagonism and this subset may dominate in cell culture, but be less prominent in the intact brain). In addition, on-site accumulation of desmoteplase in vivo may be restricted because of limited access or faster intracerebral distribution.
Because we have previously shown that rtPA exacerbates NMDA receptor-mediated neuronal death by binding to and cleaving the NR1 subunit of NMDA receptor,23 our results suggest that desmoteplase might not be a ligand for NMDA receptors. It is tempting to speculate that the K2 domain, present in tPA but not in desmoteplase, may play a critical role in the interaction between NR1 and tPA. However, the detrimental effects of rtPA may not be related solely to its catalytic or neurotoxic activities. For instance, exogenous rtPA decreases cerebral vascular resistance in rats24 and pigs.25 Moreover, LRP and integrins have recently been shown to mediate tPA-activation of smooth muscle cells.26 Similarly, also tPA-mediated induction of MMP 9 in cerebral microvascular cells as well as in astrocytes has been reported.10,27 In this context, tPA could also contribute to the extent of NMDA-mediated brain injury at late stages by acting through MMP9 induction at the neurovascular unit level and intraparenchymally. However in our model of NMDA-induced lesion, no hemorrhage has been observed. These data suggest that although we cannot fully exclude a deleterious vascular contribution of rtPA, the aggravation of the tPA-mediated NMDA-induced lesion is at least in part caused by the pro-excitotoxic action of this serine protease.
The data presented here indicate that both tPA and desmoteplase can traverse the BBB by way of the same receptor-mediated process (supplemental Figure I, available online at http://stroke.ahajournals.org). Altogether, these data suggest that preventing the interaction of tPA with LRP could be an interesting adjunctive strategy to diminish the potentially deleterious effects of tPA. In addition to the limit of the translocation of tPA from the circulation to the brain parenchyma, this approach should also increase its plasma half-life by limiting its hepatic clearance.28 However, this could increase the risk of hemorrhages and dose readjustment may be necessary. Yet, by virtue of its lack of neurotoxicity, desmoteplase may be a safer option in the treatment of ischemic stroke. Indeed, a Phase III clinical trial is underway to determine the clinical benefits of desmoteplase for the treatment of acute ischemic stroke.
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Acknowledgments |
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Sources of Funding
This work was supported by grants from the INSERM, Université de Caen, European Council (FEDER), FP6-project DiMI-LSHB-CT-2005-512146, Foundation Paul Hamel, Regional Council of Lower Normandy. This study was supported in part by research funding from PAION Deutschland GmbH and Forest Laboratories Inc (USA) to D.V.
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Footnotes |
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Present address for J.P.L.-A.: Instituto de Neurociencias de Alicante (UMH-CSIC), Campus de Sant Joan, Spain.
Received June 28, 2006; revision received September 15, 2006; accepted October 17, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- Hacke W, Donnan G, Fieschi C, Kaste M., von Kummer R, Broderick JP, Brott T, Frankel M, Grotta JC, Haley EC Jr, Kwiatkowski T, Levine SR, Lewandowski C, Lu M, Lyden P, Marler JR, Patel S, Tilley BC, Albers G, Bluhmki E, Wilhelm M, Hamilton S. ATLANTIS Trial Investigators; ECASS Trials Investigators; NINDS rt-PA Study Group Investigators. Association of outcome with early stroke treatment: pooled analysis of ATLANTIS, ECASS, and NINDS rt-PA stroke trials. Lancet. 2004; 363: 768–774.[CrossRef][Medline] [Order article via Infotrieve]
- The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rtPA Stroke Study Group. N Engl J Med. 1995; 333: 1581–1587.
[Abstract/Free Full Text] - Schleuning WD, Alagon A, Boidol W, Bringmann P, Petri T, Kratzschmar J, Haendler B, Langer G, Baldus B, Witt W, Donner P. Plasminogen activators from the saliva of Desmodus rotundus (common vampire bat): unique fibrin specificity. Ann NY Acad Sci. 1992; 667: 395–403.[Abstract]
- Hacke W, Albers G, Al-Rawi Y, Bogousslavsky J, Davalos A, Eliasziw M, Fischer M, Furlan A, Kaste M, Lees KR, Soehngen M, Warach S; DIAS Study Group. The Desmoteplase in Acute Ischemic Stroke Trial (DIAS): a phase II MRI-based 9-hour window acute stroke thrombolysis trial with intravenous desmoteplase. Stroke. 2005; 36: 66–73.
[Abstract/Free Full Text] - Furlan AJ, Eyding D, Albers GW, Al-Rawi Y, Lees KR, Rowley HA, Sachara C, Soehngen M, Warach S, Hacke W; DEDAS Investigators. Dose Escalation of Desmoteplase for Acute Ischemic Stroke (DEDAS): evidence of safety and efficacy 3 to 9 hours after stroke onset. Stroke. 2006; 37: 1227–1231.
[Abstract/Free Full Text] - Liberatore GT, Samson A, Bladin C, Schleuning WD, Medcalf RL. Vampire bat salivary plasminogen activator (desmoteplase): a unique fibrinolytic enzyme that does not promote neurodegeneration. Stroke. 2003; 34: 537–543.
[Abstract/Free Full Text] - Reddrop C, Moldrich RX, Beart PM, Farso M, Liberatore GT, Howells DW, Petersen KU, Schleuning WD, Medcalf RL. Vampire bat salivary plasminogen activator (desmoteplase) inhibits tissue-type plasminogen activator-induced potentiation of excitotoxic injury. Stroke. 2005; 36: 1241–1246.
[Abstract/Free Full Text] - Benchenane K, Lopez-Atalaya JP, Fernandez-Monreal M, Touzani O, Vivien D. Equivocal roles of tissue-type plasminogen activator in stroke-induced injury. Trends Neurosci. 2004; 27: 155–160.[CrossRef][Medline] [Order article via Infotrieve]
- Kaur J, Zhao Z, Klein GM, Lo EH, Buchan AM. The neurotoxicity of tissue plasminogen activator? J Cereb Blood Flow Metab. 2004; 24: 945–963.[Medline] [Order article via Infotrieve]
- Wang X, Lee SR, Arai K, Lee SR, Tsuji K, Rebeck GW, Lo EH. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med. 2003; 9: 1313–1317.[CrossRef][Medline] [Order article via Infotrieve]
- Benchenane K, Berezowski V, Ali C, Fernandez-Monreal M, Lopez-Atalaya JP, Brillault J, Chuquet J, Nouvelot A, MacKenzie ET, Bu G, Cecchelli R, Touzani O, Vivien D. Tissue-type plasminogen activator crosses the intact blood-brain barrier by low-density lipoprotein receptor-related protein-mediated transcytosis. Circulation. 2005; 111: 2241–2249.
[Abstract/Free Full Text] - Benchenane K, Berezowski V, Fernandez-Monreal M, Brillault J, Valable S, Dehouck MP, Cecchelli R, Vivien D, Touzani O, Ali C. Oxygen glucose deprivation switches the transport of tPA across the blood-brain barrier from an LRP-dependent to an increased LRP-independent process. Stroke. 2005; 36: 1065–1070.
[Abstract/Free Full Text] - Cecchelli R, Dehouck B, Descamps L, Fenart L, Buee-Scherrer V, Duhem C, Lundquist S, Rentfel M, Torpier G, Dehouck MP. In vitro model for evaluating drug transport across the blood-brain barrier. Adv Drug Deliv Rev. 1999; 36: 165–178.[CrossRef][Medline] [Order article via Infotrieve]
- Brillault J, Berezowski V, Cecchelli R, Dehouck MP. Intercommunications between brain capillary endothelial cells and glial cells increase the transcellular permeability of the blood-brain barrier during ischaemia. J Neurochem. 2002; 83: 807–817.[CrossRef][Medline] [Order article via Infotrieve]
- Bringmann P, Gruber D, Liese A, Toschi L, Krätzschmar J, Schleuning WD, Donner P. Structural features mediating fibrin selectivity of vampire bat plasminogen activators. J Biol Chem. 1995; 270: 25596–25603.
[Abstract/Free Full Text] - Tsirka SE, Gualandris A, Amaral DG, Strickland S. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature. 1995; 377: 340–344.[CrossRef][Medline] [Order article via Infotrieve]
- Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nat Med. 1998; 4: 228–231.[CrossRef][Medline] [Order article via Infotrieve]
- Lo EH, Broderick JP, Moskowitz MA. tPA and proteolysis in the neurovascular unit. Stroke. 2004; 35: 354–356.
[Free Full Text] - Godfrey KR, Tanswell P, Bates RA, Chappell MJ, Madden FN. Nonlinear pharmacokinetics of tissue-type plasminogen activator in three animal species: a comparison of mathematical models. Biopharm Drug Dispos. 1998; 19: 131–140.[CrossRef][Medline] [Order article via Infotrieve]
- Herz J, Strickland DK. LRP: a multifunctional scavenger and signaling receptor. J Clin Invest. 2001; 108: 779–784.[CrossRef][Medline] [Order article via Infotrieve]
- Orth K, Willnow T, Herz J, Gething MJ, Sambrook J. Low density lipoprotein receptor-related protein is necessary for the internalization of both tissue-type plasminogen activator-inhibitor complexes and free tissue-type plasminogen activator. J Biol Chem. 1994; 269: 21117–21122.
[Abstract/Free Full Text] - Chen ZL, Indyk JA, Bugge TH, Kombrinck KW, Degen JL, Strickland S. Neuronal death and blood-brain barrier breakdown after excitotoxic injury are independent processes. J Neurosci. 1999; 19: 9813–9820.
[Abstract/Free Full Text] - Fernandez-Monreal M, Lopez-Atalaya JP, Benchenane K, Cacquevel M, Dulin F, Le Caer JP, Rossier J, Jarrige AC, Mackenzie ET, Colloc’h N, Ali C, Vivien D. Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signalling. J Biol Chem. 2004; 279: 50850–50856.
[Abstract/Free Full Text] - Nassar T, Akkawi S, Shina A, Haj-Yehia A, Bdeir K, Tarshis M, Heyman SN, Higazi AA. In vitro and in vivo effects of tPA and PAI-1 on blood vessel tone. Blood. 2004; 103: 897–902.
[Abstract/Free Full Text] - Armstead WM, Cines DB, Higazi AA. Altered NO function contributes to impairment of uPA and tPA cerebrovasodilation after brain injury. J Neurotrauma. 2004; 21: 1204–1211.[CrossRef][Medline] [Order article via Infotrieve]
- Akkawi S, Nassar T, Tarshis M, Cines DB, Higazi AA. LRP and alphavbeta3 mediate tPA activation of smooth muscle cells. Am J Physiol Heart Circ Physiol. 2006; 291: H1351–H1359.
[Abstract/Free Full Text] - Wang S, Lee SR, Guo SZ, Kim WJ, Montaner J, Wang X, Lo EH. Reduction of tissue plasminogen activator-induced matrix metalloproteinase-9 by simvastatin in astrocytes. Stroke. 2006; 37: 1910–1912.
[Abstract/Free Full Text] - Biessen EA, van Teijlingen M, Vietsch H, Barrett-Bergshoeff MM, Bijsterbosch MK, Rijken DC, van Berkel TJ, Kuiper J. Antagonist of the mannose receptor and the LDL receptor-related protein dramatically delay the clearance of tissue plasminogen activator. Circulation. 1997; 95: 46–52.
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