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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Center for Molecular and Vascular Biology and
Center for Transgene Technology and Gene Therapy, Flanders
Interuniversity Institute for Biotechnology, KU Leuven,
Leuven, Belgium.
In the absence of arterial recanalization, thrombolytic agents
induce a dose-related extension of focal cerebral ischemic injury (FII)
in experimental animals. However, FII is smaller in mice lacking
Thrombolytic therapy of ischemic stroke with
alteplase improves clinical outcome at 3 months.1,2
However, alteplase infusion increased infarct volume after focal
cerebral ischemic injury (FII) induced by occlusion of the middle
cerebral artery (MCA) in mice,3 possibly as a result of
plasmin-mediated degradation of laminin, a component of basal lamina
playing a role in neuronal survival.4 The deleterious
effects of alteplase on FII in mice were confirmed and extended using
alteplase, streptokinase, or staphylokinase in hamsters,5
indicating that this phenomenon is neither species specific nor agent
specific and that it occurs in the absence of systemic fibrinogen
breakdown. Thus, one could hypothesize that thrombolytic therapy for
ischemic stroke might cause infarct expansion in cases with persistent
cerebral arterial occlusion and indeed be harmful to a subgroup of
patients. This would, however, imply that the clinical benefits of
alteplase in stroke were due to recanalization (which was not measured) and increases in infarct volume were due to a lack of recanalization. The development of specific conjunctive strategies to counteract the
potential deleterious effects of thrombolytic agents or of alternative
nonthrombolytic treatments appears to be warranted.
FII induced by MCA occlusion is increased in mice with a genetic
deficiency of plasminogen, a substrate of thrombolytic
agents6 and, conversely, reduced in mice with genetic
deficiency of In the present study, the effects on FII of reduction of
circulating Mice and hamsters
Human plasmin and human miniplasmin
Pli was prepared by adding lysine-Sepharose to human plasminogen (200 mg) solution. The mixture was stirred, washed, and resuspended in 0.1 M NaH2PO4 buffer, pH 7.4, and urokinase (1 µM final) was added overnight at 4°C. The gel was then washed to remove urokinase, eluted with 0.1 M NaH2PO4 buffer containing 0.05 M 6-aminohexanoic acid, glycerol was added to 10%, and the pool was dialyzed at 4°C against 0.1 M NaH2PO4 buffer containing 10% glycerol. The final recovery was 25 mL solution with a protein concentration of 2.8 mg/mL and an active plasmin concentration of 33 µM, comprising less than 1% residual plasminogen. Human miniplasminogen was prepared by digestion of purified human plasminogen with insolubilized elastase, followed by gel filtration and chromatography on lysine-Sepharose, as described elsewhere.9 Briefly, purified human plasminogen (900 mg) was dissolved in 0.1 M NH4HCO3, pH 7.8, containing 700 KIU aprotinin per milliliter. Insolubilized elastase (7.5 g wet gel) was added and the mixture stirred for 24 hours at room temperature. After removal of the elastase gel by filtration, the mixture was gel-filtered on a Sephadex G75 equilibrated with NH4HCO3, pH 7.8, and passed through a lysine-Sepharose column. The mPli was prepared by digestion of 650 µL miniplasminogen (1.1 mg/mL) dissolved in 0.05 M phosphate buffer, pH 7.4, containing 10% glycerol with insolubilized urokinase (1% final concentration) for 5 minutes at 37°C. The final solution had a protein concentration of 0.9 mg/mL and an active mPli concentration of 18.5 µM. Fab fragments of murine monoclonal antibodies neutralizing murine
 2-AP were
produced essentially as previously described10,11 but
using mice with inactivated 2-AP genes.12
The mice were immunized by subcutaneous injection of 50 µg murine
2-AP purified as described elsewhere.13
Spleen cells were isolated and fused with P3X63-Ag8.653 myeloma cells. Hybridomas secreting monoclonal antibodies were screened for
neutralizing activity against murine 2-AP. Positive
clones were used for production of ascitic fluid in pristane-primed
mice. The monoclonal antibodies were purified from ascites by affinity
chromatography on protein A-Sepharose14 and again tested
for neutralizing activity against purified murine and human
2-AP. A total of 63 hybridomas produced antibodies
against murine 2-AP, of which 14 neutralized murine 2-AP and 4 also neutralized human 2-AP.
Following purification, monoclonal antibody MAP-4H9 inhibited 60% of
murine 2-AP in equimolar mixtures.
Fab fragments were produced from 1 mg/mL solutions of MAP-4H9 by
digestion with 1% (wt/wt) papain in the presence of 50 mM cysteine and
1 mM ethylenediaminetetraacetic acid for 2 hours at 37°C. The
reaction was arrested by addition of 75 mM iodoacetamide. After
dialysis, the mixture was passed over a protein A-Sepharose column,
which binds an Fc fragment and uncleaved immunoglobulin G (IgG). From
21 mg MAP-4H9, 14 mg Fab-4H9 was obtained, which neutralized murine
The 2-antiplasmin in murine or hamster
plasma were measured by a chromogenic substrate assay based on its
rapid inhibition of plasmin.15 Briefly, 10 µL murine or
hamster plasma (diluted 1:10 in 0.05 M NaH2PO4
buffer, pH 7.4, containing 0.01% Tween 20) was mixed at 37°C with
420 µL 0.05 M Tris-HCl, 0.1 M NaCl buffer, pH 7.4, containing 0.01%
Tween 20, and with 20 µL 0.125 µM Pli (final concentration 5 nM).
After 10 seconds of incubation, 50 µL 3 mM S2403
(Chromogenics, Antwerp, Belgium) was added and the change in absorbance
measured at 405 nm. The change in absorbance is approximately 0.18 per
minute-1 with buffer and 0.09 per
minute-1 with pooled murine or hamster plasma,
which was used for the construction of a calibration curve.
Physiologic parameters Animal experiments were conducted according to the guiding principles of the American Physiological Society. Hemodynamic parameters were measured in mice and hamsters under anesthesia with 1 mL/kg of a mixture of ketamine (75 mg/mL; Apharmo, Arnhem, The Netherlands) and xylazine (5 mg/mL; Bayer, Leverkusen, Germany).Blood pressure was measured by insertion of a pressure transducer (SPR-671, Millar Instruments, Houston, TX) in the left carotid artery before and during 15 minutes after injection of 0.2 mg Pli in mice or 1 mg Pli in hamsters. Blood pH, PCO2, PO2, and hemoglobin (Hb) concentration were measured using a blood gas analyzer (ABL4, Radiometer, Copenhagen, Denmark) before and 15 minutes after injection of saline or 0.2 mg Pli in mice or 1 mg Pli in hamsters. Murine cerebral ischemic infarction model FII was produced by persistent occlusion of the MCA, as described in detail elsewhere.6,16 Anesthesia was routinely performed with 1 mL/kg of a mixture of ketamine (75 mg/mL; Apharmo) and xylazine (5 mg/mL; Bayer). Alternatively, to establish that these drugs did not affect FII size, anesthesia was performed with inhalation of 2% isoflurane in oxygen. During the surgery and until recovery from anesthesia, the animals were kept on a heated pad that was maintained at 37 ± 0.5°C.The following experimental groups of BALB/c mice were studied: (1) Pli as a bolus intravenous dose of 0.07 mg or 0.2 mg either 15 minutes before or 15 minutes after ligation of the MCA; (2) mPli as a bolus of 0.1 mg or 0.2 mg 15 minutes before ligation of the MCA; or (3) Fab-4H9 as a bolus of 1 mg given 15 minutes before or 1.7 mg given 15 minutes after MCA ligation. Each experimental group was matched by a saline control group of equal size. To determine whether plasmin activity was necessary to affect FII, 0.2 mg Pli, inactivated with 1500 KIU aprotinin (Trasylol, Bayer, Leverkusen, Germany) that had less than 1% residual plasmin activity, was given after 15 minutes of ligation of the MCA and compared with groups receiving Pli or aprotinin alone. Pli at a dose of 0.07 mg was given to BALB/c, C57Bl/6, or SV129 mice to evaluate the effect of genetic background. The animals were allowed to recover and were then returned to their
cages. After 24 hours, the mice were anesthetized and a blood sample
was taken by heart puncture to determine Immunohistochemical analysis Immunostaining for laminin and for fibrinogen was carried out in mice given saline or 0.2 mg Pli 15 minutes after MCA occlusion, followed by perfusion with 1% paraformaldehyde in PBS at 24 hours. As a control, mice with stereotactic injection (Model 900, David Kopf Instruments, Tujunga, CA) of 1.5 nM kainic acid in the CA1 region in the hippocampus,19 followed by perfusion with 1% paraformaldehyde in PBS at 24 hours, were used. The coordinates of the injection were as follows: bregma 2.0 mm, mediolateral 1.3 mm,
dorsoventral 1.5 mm.
Brains were removed, embedded in paraffin, and sectioned in 5 µm slices. For immunostaining of laminin, sections were incubated with a primary rabbit antilaminin antibody (Sigma Chemical, St. Louis, MO) diluted 1:50, followed by a peroxidase-labeled swine antirabbit IgG (Dako, Glostrup, Denmark) diluted 1:50. Fibrin(ogen) was stained via a 3-step procedure with a goat antimouse fibrinogen (Nordic Immunologies, Tilburg, The Netherlands) diluted 1:200, followed by rabbit antigoat IgG (Dako) diluted 1:100 and goat peroxidase-antiperoxidase complex (Dako) diluted 1:50. Peroxidase activity was developed by incubating sections in 0.05 M Tris-HCl buffer, pH 7.0, containing 0.06% 3,3'-diaminobenzidine and 0.01% H2O2 with enhancement of laminin staining by 7 mM ammonium nickel(II) sulfate. Counterstaining with hematoxylin was used to identify neuronal atrophy. Hamster FII model FII was produced by a combination of persistent occlusion of the left MCA and the left common carotid artery (CCA) and a transient occlusion of the right CCA, as described in detail elsewhere.5 The MCA was occluded by ligation with 10-0 nylon thread (Ethylon, Neuilly, France), after which the artery was transsected distally and the temporal muscle and skin were sutured back in place. A vertical incision was then made above the sternum, and both CCAs were exposed. The left CCA was ligated with 2 7-0 nylon threads and transsected in between. The right CCA was occluded with an arterial clamp during 30 minutes and then released. During the clamping, an incision was made in the right groin, and the femoral vein was cannulated with a 2FG catheter for study drug administration. Finally, the skin wound was closed. Hamsters were given a saline or 1 mg Pli bolus injection 15 minutes after release of the right CCA occlusion. The animals were then allowed to recover and were returned to their cages. After 24 hours, the animals were killed with an overdose of Nembutal (500 mg/kg; Abbott) and decapitated. The brain was removed and processed for planimetry as described above. One animal in the saline group died during the 24 hours observation period and was omitted from the analysis.Statistical analysis The data are represented as median and range (for cerebral infarct size) or as mean ± SEM (for coagulation and hemodynamic parameters) of the number of determinations. The significance of differences was determined using Mann-Whitney test or Student t test, as appropriate.
Effects of injection of human plasmin on hemodynamic variables Bolus intravenous injection of 0.2 mg Pli in mice or 1 mg Pli in hamsters did not affect arterial pH, PCO2, PO2, Hb concentration, or arterial blood pressure (Table 1).
Effects of human plasmin or Fab-4h9 on
2-AP and fibrinogen levels are summarized in Table 2. In all
experimental groups, 2-AP levels had normalized within
24 hours, whereas levels of fibrinogen, an acute phase reactant, had
increased to 1.2 to 1.9 g/L. Thus, 2-AP depletion was
transient and only persisted during the first hours after the single
intravenous bolus injection of the depleting agent.
Injection of 1 mg Pli in hamsters depleted the Effect of human plasmin on FII in mice Ligation of the MCA induced FII with a volume of 28 µL (range, 20-34) (n = 10) in inbred BALB/c mice (Figure 1A), 6.9 µL (range, 0.3-9.0) (n = 8) in SV129 mice, and 15 µL (range, 8.0-23) (n = 12) in C57BL/6 mice (Table 3). Injection of 0.07 mg Pli in BALB/c mice reduced FII to 23 µL (range, 17-26) (n = 9, P = .01 vs saline). Similar decreases were observed whether the Pli injection was given 15 minutes before or 15 minutes after ligation of the MCA. In C57Bl/6 mice, injection of 0.07 mg Pli reduced FII to 11µL (range, 1.8-16) (n = 12, P = 0.008 vs saline). In SV129 mice, with much smaller infarcts, injection of 0.07 mg Pli produced a similar 30% reduction of FII. Injection of 0.2 mg Pli in BALB/c mice either 15 minutes before or 15 minutes after MCA ligation induced a 20% reduction of FII (Table 3). Injection of 0.1 mg mPli in BALB/c mice reduced FII after MCA ligation from 26 µL (range, 20-27) (n = 5) to 24 µL (range, 18-25) (n = 6, P = .14), whereas 0.2 mg mPli reduced FII from 27 µL (range, 23-32) (n = 6) to 20 µL (range, 13-26) (n = 6, P = .025). Injection of a mixture of 0.2 mg Pli and 1500 KU aprotinin, with a residual plasmin activity of less than 1%, produced a FII volume of 28 µL (range, 25-30) (n = 6) indistinguishable from that of aprotinin alone, at 29 µL (range, 24-30) (n = 6), whereas the fibrinogen and2-AP levels measured after 15 minutes (when excess aprotinin was cleared from the
circulation) remained unchanged (data not shown).
The relative contribution of necrosis and edema to ischemic injury was determined by integration of the infarct areas; the necrotic areas, ie, the difference between the contralateral and the ipsilateral viable areas; and the edema areas, ie, the difference between infarct and necrotic areas (Table 3). In control BALB/c mice, the infarct volume of 31 µL (range, 27-32) consisted of 26 µL (range 22-27) necrotic volume and of 4.6 µL (range 1.4-5.8) edema, representing 14%. In mice given 0.2 mg Pli, the infarct volume of 26µL (range 24-28) (P = .01 vs saline) consisted of 22 µL (range 20-26) necrotic volumes (P = .025 vs saline) and of 3.4 µL (range 0.0-5.9) edema (P = .33 vs saline). FII in BALB/c mice anesthetized with isoflurane was 28 µL (range 27-31) (n = 6) with saline and 26µL (range 20-27) (n = 6) with 0.2 mg Pli (P = .006). Effect of Fab-4H9 on cerebral infarct size in mice Injection of 1 mg of Fab-4H9 15 minutes before MCA ligation reduced FII from 26 µL (range 21-27) (n = 6) to 22 µL (range 15-24) (n = 6, P = .055) (Table 2). Injection of 1.7 mg Fab-4H9 15 minutes after MCA ligation reduced FII from 28 µL (range 25-30) (n = 6) to 24 µL (range 20-27) (n = 6, P = .032).Effect of human plasmin on cerebral infarct size in hamsters Injection of 1 mg Pli after release of the transient occlusion of the right CCA reduced FII from 20 µL (range 9.9-38) (n = 6) with saline (Figure 1B, Table 2) to 7.0 µL (range 0.44-31) (n = 7) with Pli (P = .032). In control hamsters, FII of 20 µL (range 9.9-38) contained 2.2 µL (range 0.0-5.6) edema, representing 12%. The reduction in FII was primarily due to reduction of necrosis from 16 µL (range 9.9-33) with saline to 4.6 µL (range 0.44-26) with Pli (P = .015) and not to differences in edema (Table 4).
Immunohistochemical staining Light microscopic analysis of brain sections of the contralateral cerebral cortex (Figure 2) stained for hematoxylin revealed normal neurons (Figure 2A) surrounded by laminin immunoreactivity (Figure 2D) both in saline- and in Pli-treated mice. In the FII zone, neuronal atrophy was observed (Figure 2B,C), and immunoreactivity of laminin was slightly but not significantly reduced and condensed around neurons (Figure 2E,F). No difference was observed between saline-treated animals (Figure 2B,E) and Pli-treated animals (Figure 2C,F). Laminin immunoreactivity of blood vessels in the infarct area was very similar among saline-treated mice (Figure 2E) and Pli-treated mice (Figure 2F) and comparable to that of the contralateral area (Figure 2D). In the CA1 region in the hippocampus, a similar change in laminin staining was observed by injection of kainic acid (Figure 2G) relative to the contralateral side (Figure 2H). Fibrin(ogen) immunoreactivity (brown staining) was not observed in the contralateral area (Figure 2A) but was comparably present in the infarct area of saline-treated animals (Figure 2B) and Pli-treated animals (Figure 2C).
Thrombolytic therapy for ischemic stroke is based on the premise that timely recanalization of the occluded cerebral artery may salvage "the ischemic penumbra,"20 the hypoperfused but potentially viable zone adjacent to the central ischemic area, limit infarct size, and improve functional recovery and survival. Early thrombolysis with alteplase indeed restored reperfusion, salvaged jeopardized brain tissue, and limited cerebral infarct size in experimental animals21 and reduced morbidity and mortality in patients.1,2 Studies in experimental animal models,3,4 however, indicate that at least in cases with persistent occlusion there may be deleterious side effects of the thrombolytic agent, causing infarct expansion. To the extent that extrapolation of these findings to thrombolytic therapy of ischemic stroke is warranted, this would suggest that the recently observed beneficial clinical outcome with alteplase1,2 versus the detrimental outcome with streptokinase22-24 might relate to the higher efficacy for arterial recanalization of alteplase. Because it appears to be impossible to distinguish a priori between patients who will or who will not achieve cerebral arterial recanalization with alteplase, the development of specific conjunctive strategies to counteract the harmful effects of thrombolytic agents on persisting focal cerebral ischemia appears to be warranted. In view of the interactive effects on neuronal degeneration between oxidative stress, excitotoxin induction,25 and with thrombolytic agents, one could speculate that oxygen radical scavengers, glutamate antagonists,26 or both might beneficially affect the clinical outcome of thrombolytic therapy for ischemic stroke. Alternatively, gene deficiency of plasminogen increased FII volume, and
a positive correlation was found between infarct size and
In the present study, the hypothesis that reduction of
A mouse of 30 g has an estimated total body pool of
FII may induce cerebral edema, which in turn may exacerbate ischemic damage. Determination of the relative contribution of necrosis and edema to infarct size, however, indicated that plasmin caused alterations of necrosis and not of edema. The hypothesis that FII reduction was the result of Cerebral infarct expansion mediated by tissue-type plasminogen
activator is thought to occur via degradation of laminin that surrounds
neurons, resulting in enhanced excitotoxin-mediated neuronal cell
death.4 In the present study, laminin immunoreactivity around neurons in the infarct area seemed to be somewhat reduced at 24 hours, but to a similar extent in saline- and Pli-treated animals,
indicating that the observed effect could not be ascribed to
alterations in laminin turnover. It is possible that reduction of
The concentration of
Submitted January 10, 2000; accepted January 4, 2001.
Supported in part by a sponsored research agreement between the Center for Molecular and Vascular Biology and Thromb-X, NV, a spin-off company of the University of Leuven in which D.C. has an equity interest.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: D. Collen, Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Campus Gasthuisberg O&N, Herestraat 49, B-3000 Leuven, Belgium; e-mail: desire.collen{at}med.kuleuven.ac.be
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A. Shuaib, C. Xu Wang, T. Yang, and R. Noor Effects of Nonpeptide V1 Vasopressin Receptor Antagonist SR-49059 on Infarction Volume and Recovery of Function in a Focal Embolic Stroke Model Stroke, December 1, 2002; 33(12): 3033 - 3037. [Abstract] [Full Text] [PDF]
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P. A. Lapchak, D. M. Araujo, S. Pakola, D. Song, J. Wei, and J. A. Zivin Microplasmin: A Novel Thrombolytic That Improves Behavioral Outcome After Embolic Strokes in Rabbits Stroke, September 1, 2002; 33(9): 2279 - 2284. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
2-antiplasmin by
intravenous plasmin or immunoneutralization reduces focal cerebral
ischemic injury in the absence of arterial recanalization
Top
Abstract
Introduction
/
the Fab fragment of a murine
monoclonal antibody neutralizing murine