Reversing the deleterious effects of α2-antiplasmin on tissue plasminogen activator therapy improves outcomes in experimental ischemic stroke

Abstract

High blood levels of α2-antiplasmin have been associated with failed tissue plasminogen activator (TPA) therapy for ischemic stroke. Yet, other data suggests that α2-antiplasmin may be protective in stroke, because it defends against bleeding and excitotoxicity. To address this paradox, we examined the effects of high α2-antiplasmin levels and α2-antiplasmin inactivation in mice treated with TPA 0.5-2.5h after middle cerebral artery (MCA) thromboembolism. Brain infarction, swelling, hemorrhage, blood brain barrier breakdown and neuronal apoptosis were measured by a blinded observer. Thrombus dissolution was determined by gamma counting. During TPA treatment, high α2-antiplasmin blood levels increased brain infarction (2.2-fold) and swelling (3.7-fold), but decreased MCA thrombus dissolution. Conversely, α2-antiplasmin inactivation during TPA treatment reduced brain infarction, hemorrhage and swelling, but increased MCA thrombus dissolution. Inactivation of α2-antiplasmin during TPA treatment reduced neuronal apoptosis and blood brain barrier breakdown. Inactivation of α2-antiplasmin also reduced short-term mortality. Taken together these data show that α2-antiplasmin opposes the effects of TPA therapy and contributes to enhanced brain injury after experimental thromboembolic stroke. Conversely, α2-antiplasmin inactivation during TPA treatment improves thrombus dissolution and reduces brain infarction, swelling and hemorrhage. Consistent with clinical observations, these data suggest that α2-antiplasmin exerts deleterious effects that reduce the efficacy and safety of TPA therapy for ischemic stroke.

Keywords: Apoptosis; Edema; Fibrinolysis; Hemorrhage; Mortality; Stroke; Thrombosis.

Copyright © 2014 Elsevier Inc. All rights reserved.

Figures

Figure 1

Increased a2AP levels (↑a2AP) worsen infarction and brain swelling after TPA therapy. Effects of increased a2AP levels and TPA treatment on A) brain infarction, B) brain swelling, C) dissolution of the culprit thromboembolus and D) brain hemorrhage. Mice with thromboembolic stroke were treated with TPA (10 mg/kg) after 1 h of ischemia; a2AP levels were increased by a2AP infusion immediately after thromboembolism. Mice were analyzed 6 h after thromboembolic stroke. The percent infarction was determined at the completion of the experiment by TTC-staining of serial brain slices followed by digital imaging analyses by a blinded observer. The percent swelling was determined at the completion of the experiment by digital imaging analyses of serial brain slices by a blinded observer. The percent dissolution was determined by gamma scintillation counting of the residual 125I-fibrin in the thrombus. The percent hemorrhage was determined at the completion of the experiment by digital imaging analyses of serial brain slices by a blinded observer. Data represent mean ± standard errors. N= 5 per group; ***p<0.001 vs. TPA

Figure 2

Standard dose TPA treatment with a2AP-inactivation affects infarction, dissolution of the culprit MCA thrombus and hemorrhage by comparison to standard dose TPA alone. Effects on A) brain infarction, B) dissolution of the culprit MCA thrombus and C) brain hemorrhage. Mice were treated with TPA (10 mg/kg) with or without a2AP inactivation 2.5 h after MCA thromboembolism and were analyzed 6 h after stroke. The percent hemorrhage was determined by imaging of brain slices at the completion of the experiment; similarly infarction was determined at the completion of the experiment by TTC staining of serial brain slices. Images were subjected to digital analysis by a blinded observer. Percent thrombus dissolution was determined as above. Data represent mean ± standard errors. N= 5 per group; *p

Figure 3

Low dose TPA with a2AP inactivation improves stroke outcomes vs. low dose TPA alone. Effects on A) brain infarction, B) dissolution of the culprit MCA thrombus and C) brain hemorrhage. Mice were treated with TPA (2 mg/kg) with or without a2AP inactivation 2.5 h after MCA thromboembolism and were analyzed 6 h after stroke. The percent hemorrhage was determined by imaging of brain slices at the completion of the experiment; similarly infarction was determined at the completion of the experiment by TTC staining of serial brain slices. Images were subjected to digital analysis by a blinded observer. Percent thrombus dissolution was determined as above. Data represent mean ± standard errors. N= 5 per group; *p

Figure 4

Treatment with low dose TPA with a2AP inactivation reduces mortality, brain infarction, hemorrhage and swelling vs. standard dose TPA alone. Mice were treated 30 min after MCA thromboembolism with TPA (10 mg/kg) or TPA (2 mg/kg) with a2AP inactivation. Survival was monitored and brains of mice surviving at least 12 h were analyzed. A) Effect on 24 h survival. B) Effect on brain infarction in surviving mice. Percent infarction was determined by TTC staining as described above. C) Effect on brain swelling as described above. D) Effect on hemorrhage as described above. Data represent mean ± standard errors. Survival analyses N =17, otherwise N= 5 per group. *p<0.05, **p<0.01, vs. TPA alone.

Figure 5

TPA treatment with a2AP inactivation decreases breakdown of the blood brain barrier and apoptosis indicated by TUNEL-staining. A) TUNEL staining (green) around the area of collagen IV -stained blood vessels (red). DAPI-stained nuclei are in blue. Graph, image quantification of TUNEL positive non-blood vessel cells as described in Methods. B) Detection of breakdown of the blood brain barrier by leakage of albumin (red) outside of collagen IV-stained blood vessels (green) with DAPI-stained nuclei stained in blue. In mice treated with TPA + a2AP-I, albumin (red) co-localized with collagen IV-stained blood vessels as indicated by yellow coloration in the merged image. In mice treated with TPA there was significant leakage of albumin outside collagen IV-stained blood vessels as shown by the diffuse red staining and the minimal yellow coloration in the merged image. Graph, image quantification of blood brain barrier leakage of albumin described in Methods. Data represent mean ± standard errors. N= 4 per group; **p