Intravenous Administration of a GPIIb/IIIa Receptor Antagonist Extends the Therapeutic Window of Intra-Arterial Tenecteplase–Tissue Plasminogen Activator in a Rat Stroke Model
Background and Purpose— Occlusion of the middle cerebral artery triggers platelet accumulation at the site of occlusion and in downstream microvessels. The platelet-induced secondary thrombosis promotes the progressive development of ischemic brain damage and contributes to the resistance to thrombolysis and to the tight 3-hour therapeutic window. We tested the hypothesis that combination of intravenous (IV) administration of a GPIIb/IIIa receptor antagonist, 7E3 F(ab′)2, with intra-arterial (IA) administration of tenecteplase–tissue plasminogen activator (TNK-tPA) increases the efficacy of thrombolysis and extends the therapeutic window of stroke.
Methods— Rats subjected to embolic stroke were treated with IV 7E3 F(ab′)2 (6 mg/kg) in combination with IA or IV TNK-tPA (5 mg/kg) at 4 and 6 hours after onset of stroke, respectively; IA TNK-tPA (5 mg/kg) alone at 6 hours after onset of stroke; or saline at 6 hours after onset of stroke.
Results— The combination of IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours) significantly (P<0.05) reduced infarct volume and improved neurological functional deficits, which was associated with significant (P<0.05) reductions in the size of embolus at the origin of the occluded middle cerebral artery and in down-stream microvascular platelet and fibrin deposition, and enhanced microvascular patency compared with saline-treated rats. However, combination of IV 7E3 F(ab′)2 (4 hours) and IV TNK-tPA (6 hours) or IA TNK-tPA (6 hours) alone failed to reduce infarct volume and improve neurological function compared with the saline-treated rats. No significant differences of the incidence of hemorrhage were detected among groups.
Conclusions— These data suggest that the combination of IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours) extends the therapeutic window of thrombolysis to 6 hours after stroke.
Intravenous (IV) administration of tissue-type plasminogen activator (tPA) within 3 hours of stroke onset improves outcome after acute ischemic stroke.1 However, delayed IV administration of tPA (>3 hours) fails to show therapeutic benefit and significantly increases the risk of intracerebral hemorrhage.2 Only 2% of the ischemic stroke population in the US are treated with a thrombolytic agent.3 Alternative approaches which could achieve more rapid and complete thrombolysis and yet reduce the risk of hemorrhage are needed.
Intra-arterial (IA) administration of thrombolytic agents has been used to amplify the thrombolytic effect after stroke.4 Data from the Pro-urokinase in Acute Cerebral Thromboembolism (PROACT) II trial demonstrate that IA administration of recombinant prourokinase within 6 hours of onset of stroke accelerated recanalization and improved 90-day clinical outcome.5 A recent clinical trial, the Interventional Management of Stroke (IMS) study, demonstrated that patients treated with a combined IV and IA tPA within 3 hours of onset of stroke achieved a significantly improved outcome at 3 months compared with patients treated with placebo in an historical control group.6 This study included patients who had major occlusions of the carotid artery in addition to distal occlusion in the middle cerebral artery (MCA), suggesting that a combined IV/IA approach could provide better outcome for patients with a large thrombus burden.6
Treatment strategies, which inhibit platelet aggregation, can amplify the efficacy of thrombolytic therapy.7 Adjunctive thrombolysis with tPA and 7E3 F(ab′)2, a potent GPIIb/IIIa antagonist, significantly enhanced downstream cerebral microvascular integrity and patency, reduced cerebral infarction, and improved neurological function.7 A randomized dose-escalation study shows that IV administration of abciximab within 24 hours of onset of stroke improves patient outcome at 3 months.6 Moreover, experimental studies of stroke indicated that administration of 7E3 F(ab′)2 effectively reduced brain infarction when treatment was initiated within a 3-hour therapeutic window in rats after stroke.8
Tenecteplase (TNK)-tPA is a third generation thrombolytic agent which has a longer half life, improved fibrin specificity, and increased resistance to plasminogen activator inhibitor 1 (PAI-1) as compared with wild-type tPA.9,10 Treatment of rats subjected to embolic MCA occlusion with IA TNK-tPA significantly reduced infarct volume when administered at 2 but not 4 hours after stroke onset. Accordingly, in this study we tested the hypothesis that a combined IV and IA administration of 7E3 F(ab′)2 and TNK-tPA, respectively, extends the therapeutic window of thrombolysis in a rat model of embolic stroke.
Materials and Methods
Male Wistar rats (n=59; Charles River Breeding Co, Wilmington, Mass) weighing 350 to 450g were subjected to embolic middle cerebral artery occlusion (MCAo).11 Briefly, the MCA was blocked by an Evans blue labeled clot (≈1 μL).11
7E3 F(ab′)2 was given at a dose of 6 mg/kg followed by a second dose of 6 mg/kg at 12 hours after the first dose. This dosing protocol produces marked inhibition of platelet aggregation for up to 4 days.12 TNK-tPA (Genentech) was infused at a dose of 1.5 mg/kg (10% bolus and the remainder at a continuous infusion over a 30-minute interval using a syringe infusion pump; Harvard Apparatus). This dose of TNK-tPA effectively increased recanalization rates and reduced infarct volume in stroke rats.12,13 After MCAo, animals were randomly assigned groups. To examine the effects of a combined IV and IA approach on stroke, 7E3 F(ab′)2 was administered (IV) at 4 hours and TNK-tPA was administered (IA) 6 hours after stroke (n=14). Stroke rats receiving IV administration of 7E3 F(ab′)2 and TNK-tPA at 4 and 6 hours, respectively, after onset of stroke were used as a control group (n=14). Additional control groups included IA administration of TNK-tPA alone 6 hours after stroke (n=14) and IV administration of saline 4 hours after stroke (n=17).
Measurements of Infarct Volume
Rats (n=8 per group in the combination treatment groups and TNK-tPA alone group; n=11 per group in the saline-treated group) were euthanized 7 days after MCAo, and infarct volume was measured on hematoxylin and eosin (H&E)–stained 7 coronal sections, as previously described.12 The ischemic volume is presented as the percentage of infarct volume of the contralateral hemisphere.11
Measurements of Hemorrhage
Gross hemorrhage, defined as blood evident to the unaided eye on the H&E–stained coronal sections, was evaluated on 7 coronal sections for each animal 7 days after MCAo. Gross hemorrhagic rate is presented as the percentage of animals per group with identified gross hemorrhage on any coronal section. Petechial hemorrhage, defined as a cluster of red blood cells outside of the lumen of blood vessels, was measured on 7 H&E–stained coronal sections according to the published method.
Neurological severity scores (NSS), a composite of motor, sensory, reflex, and balance tests,14 were measured 1 and 7 days after MCAo. Neurological function was graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18).
Quantitative Measurements of Embolus
To quantify the change of an embolus, we measured the Evans blue labeled clot which emits red fluorochrome at the origin of the MCA 24 hours after stroke using a fluorescent microscope. Briefly, fluorescence within the right intracranial segment of the internal carotid artery (ICA) and the origin of the MCA was digitized using the MicroComputer Imaging Device system (Imaging Research). The area of Evans blue (μm2) was calculated by tracing the areas on the computer screen, and a summation of the area of Evens blue from the image is presented as the total area of embolus.
Measurements of Microvascular Patency
To examine patency of cerebral microvessels, fluorescein isothiocyanate (FITC) dextran (2×106 molecular weight, Sigma, 1 mL of 50 mg/mL) was administered intravenously to the rats 24 hours after stroke. Three coronal sections (100 um) at bregma −0.2, −0.8, and −2.8 mm, which encompass the entire territory supplied by the MCA from each rat, were digitized and analyzed using the MicroComputer Imaging Device system.7 Briefly, 10 fields of view (4.4 mm2) from each coronal section were acquired in the territory supplied by the right MCA. To quantify microvasculature FITC-dextran perfusion, a threshold of light intensity, which detects all FITC pixels, was applied to each digitized image to ensure that the numbers of FITC pixels reflected the original FITC-dextran perfused patterns. Data are presented as the numbers of FITC pixels divided by the total numbers of pixels within the field of view, expressed as a percentage.
Immunohistochemistry and 3D Quantitation
A rabbit polyclonal antibody against rat thrombocyte (Inter-Cell Technologies Inc, Hopewell, NJ) was used at a titer of 1:200 to detect platelets. Single immunofluorescence labeling was performed to measure fibrin deposition. For quantification of fibrin immunoreactivity, 10 thin optical sections along the z axis with 1-μm step size were acquired for each of 4 fields of view from the ischemic area and contralateral homologous area in each section using a Bio-Rad MRC 1024 laser-scanning confocal imaging system (Bio-Rad).15 The fibrin(ogen) immunoreactivity in tissue samples was quantified as previously described.15
All values are presented as mean±SD. Two-way ANOVA was used to test overall treatment effects of ordinal data between groups. Fisher exact test was used to test the gross hemorrhagic rates among the groups. Statistical significance was set at P<0.05.
Infarct Volume and Neurological Outcome
Stroke rats treated with IV 7E3 F(ab′)2 and IA TNK-tPA at 4 and 6 hours after stroke onset, respectively, exhibited a significant (P<0.05) reduction of infarct volume (Figure 1) and an improvement of neurological outcome measured by NSS (Figure 2) compared with rats in saline-treated group 7 days after stroke. In contrast, IV combination of 7E3 F(ab′)2 (4 hours) and TNK-tPA (6 hours) or monotherapy with IA TNK-tPA (6 hours) failed to reduce infarct volume (Figure 1) and improve NSS (Figure 2) compared with the saline control group. No animal deaths were found.
Gross hemorrhage in the ipsilateral lesion was detected in 25% of the rats treated with combination of IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours), 13% of combination of IV 7E3 F(ab′)2 (4 hours) and IV TNK-tPA (6 hours), 38% of TNK-tPA–treated group (IA, 6 hours), and 18% of saline control group. Petechial hemorrhage was 76.2±123.6 μm2 in rats treated with combination of IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours), 51.4±78.6 μm2 in combination of IV 7E3 F(ab′)2 (4 hours) and IV TNK-tPA (6 hours), 115.9±181.1 μm2 in the TNK-tPA–treated group (IA, 6 hours), and 38.3±60.6 μm2 in the saline-treated group. However, there were no statistically significant differences of gross and petechial hemorrhage among the groups.
Measurements of an Embolus and Cerebral Vessel Patency
To examine the embolus and the patency of cerebral vessels after cerebral ischemia, Evans blue labeled clots were used to induce cerebral ischemia, and FITC-dextran was injected 5 minutes before animals were euthanized. A large portion of Evans blue dye was detected within the intracranial segment of the right ICA and the origin of the MCA where little FITC-dextran was detected 24 hours after MCA occlusion in rats (n=3) treated with saline (Figure 3). However, rats (n=3) treated with the combination of IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours) showed a significant (P<0.05) reduction in the area with Evans blue compared with rats treated with saline (Figure 3). These rats also had significantly (P<0.05) larger areas of downstream microvessels perfused by FITC-dextran than rats in the saline-treated group (Figure 4A). Rats (n=3 per group) treated with IA TNK-tPA alone (6 hours) and with combination of IV 7E3 F(ab′)2 (4 hours) and IV TNK-tPA (6 hours) did not exhibit a significant reduction in embolus area at the origin of the MCA (Figure 3), and the downstream microvasculature remained plasma deficient (Figure 4A).
Platelet and Fibrin Deposition
Combination treatment with IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours) significantly (P<0.05) reduced the number of microvessels with platelet aggregation (Figure 4B) and the volume of intravascular fibrin deposition (Figure 5) in the ipsilateral hemisphere compared with saline-treated rats. However, treatment with IA TNK-tPA (6 hours) alone and with combination of IV 7E3 F(ab′)2 (4 hours) and IV TNK-tPA (6 hours) failed to reduce the number of microvessels with platelet aggregation (Figure 4B) and the volume of intravascular fibrin deposition (Figure 5) compared with saline-treated rats.
In this study, we demonstrated that combination of IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours) significantly reduced infarct volume and improved neurological function, which was associated with significant reductions in the embolus at the origin of the occluded MCA and in the down-stream microvascular perfusion deficits without increasing the incidence of hemorrhagic transformation. Therefore, our data suggest that combined IV administration of a GPIIb/IIIa antagonist with IA administration of a fibrinolytic agent extends the therapeutic window for thrombolysis. The neuroprotection of the combination treatment is presumably because of the enhancement of thrombolysis which improves patency of cerebral vessels after stroke.
Stroke activates platelets.16,17 When a single embolus is placed at the origin of the MCA, platelets accumulate at the site of the embolus occluding the MCA with the time.16 The contents of the embolus are changed from a fibrin-rich clot to a composition of fibrin, platelets, and leukocytes.16 Occlusion of the MCA also triggers platelet-mediated thrombosis in downstream microvessels.16 Activated platelets retract clot and the release of plasminogen activator inhibitor 1, α 2–antiplasmin, and factor XIII, all of which contribute to resistance to fibrinolysis.18,19 Consistent with these findings, our data demonstrate that TNK-tPA (IA, 6 hours) alone resulted in minimal recanalization at the origin of the MCA, although TNK-tPA is relatively resistant to PAI-1 compared with tPA.10 In contrast, the combination of IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours) significantly increased the recanalization rate after embolic stroke and reduced platelet as well as fibrin deposition within downstream microvessels, indicating that the reduction of the embolus at the site of the occlusion improves downstream cerebral microvascular patency.7 In addition, the combination of IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours) significantly reduced infarct volume and improved functional outcome, whereas the combination of IV 7E3 F(ab′)2 (4 hours) and IV TNK-tPA (6 hours) failed to reduce infarct volume and improve neurological outcome. The therapeutic window for the treatment of ischemic stroke with IV tPA is 3 hours,1 although a recent study suggests a potential benefit beyond 3 hours for milder strokes.20
By demonstrating that a combination of IV/IA approach is superior to a combination of IV/IV for lengthening the thrombolytic therapeutic window to 6 hours after stroke, the present data extend our previous study that IV tPA and 7E3 F(ab′)2 effectively reduced cerebral infarction when administered at 4 hours after stroke onset, whereas IV 7E3 F(ab′)2 alone at 4 hours after stroke onset failed to provide any therapeutic benefit.7 The site of occlusion is an important variable of thrombolytic therapy.21 In our model of embolic focal ischemia, the embolus is placed at the origin of the MCA, which is the most difficult condition for thrombolysis.22 The potential advantages of IA thrombolysis are increased local thrombolytic concentration and hence improved recanalization rates, and subsequently a lower risk of cerebral hemorrhage due to a lower systemic exposure to the thrombolytic agent.23 To optimize the treatment, a dose-response study to investigate IA TNK-tPA in combination with 7E3 F(ab′)2 is warranted. Consistent with our results, a recent clinical trial, the IMS study, demonstrated that patients who had major occlusions of the carotid artery in addition to distal occlusion in the MCA treated with combined IV/IA tPA within 3 hours of onset of stroke achieved a significantly better outcome at 3 months than patients treated with placebo in the histological control group.6 Other clinical studies also indicate that recanalization even at 1 day after symptom onset is associated with a reduced lesion growth and a favorable clinical course compared with persisted occlusion of the MCA.24,25
Thrombolysis is associated with a markedly increased risk of hemorrhagic transformation, which is one of the major obstacles of the clinical use of thrombolytic therapy.1,21 In addition, reperfusion after delayed tPA treatment could damage the cerebral microvasculature and exacerbate ischemic cell damage and promote hemorrhagic transformation.26,27 The finding that the combination of IV 7E3 F(ab′)2 (4 hours) and IA TNK-tPA (6 hours) significantly reduced microvessel platelet deposition, improved microvascular patency, and reduced cerebral infarction without increasing the incidence of hemorrhagic transformation suggests that the platelet plays an important role in the reperfusion injury, which is consistent with previous studies.7,28
In conclusion, the present study demonstrates that treatment of embolic stroke with IV 7E3 F(ab′)2 (4 hours) in combination with IA TNK-tPA (6 hours) significantly reduced infarct volume and improved functional outcome without increasing the incidence of hemorrhagic transformation. The neuroprotection of the combination treatment is presumably because of the enhancement of thrombolysis which improves patency of cerebral vessels after stroke.
This work was supported by National Institute of Neurological Disorders and Stroke (NINDS) grants PO1 NS23393, PO1 NS42345, RO1 HL64766, and RO1 NS38292. We thank Cynthia Roberts, Xuepeng Zhang, and Qing-e Lu for technical assistance.
- Received August 26, 2004.
- Accepted September 17, 2004.
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