Annexin A2 combined with low-dose tPA improves thrombolytic therapy in a rat model of focal embolic stroke - PubMed (original) (raw)

Annexin A2 combined with low-dose tPA improves thrombolytic therapy in a rat model of focal embolic stroke

Haihao Zhu et al. J Cereb Blood Flow Metab. 2010 Jun.

Abstract

Recent studies showed that soluble annexin A2 dramatically increases tissue plasminogen activator (tPA)-mediated plasmin generation in vitro, and reduces thrombus formation in vivo. Here, we hypothesize that combining annexin A2 with tPA can significantly enhance thrombolysis efficacy, so that lower doses of tPA can be applied in ischemic stroke to avoid neurotoxic and hemorrhagic complications. In vitro activity assays confirmed tPA-specific amplification of plasmin generation by recombinant annexin A2. In a rat focal embolic stroke model, combination therapy with tPA and recombinant annexin A2 protein at 2 h post-ischemia decreased the effective dose required for tPA by four-fold and reduced brain infarction. Combining annexin A2 with tPA also lengthened the time window for thrombolysis. Compared with tPA (10 mg/kg) alone, the combination of annexin A2 (5 mg/kg) plus low-dose tPA (2.5 mg/kg) significantly enhanced fibrinolysis, attenuated mortality, brain infarction, and hemorrhagic transformation, even when administered at 4 h post-ischemia. Combination with recombinant annexin A2, the effective thrombolytic dose of tPA can be decreased. As a result, brain hemorrhage and infarction are reduced, and the time window for stroke reperfusion prolonged. Our present findings provide a promising new approach for enhancing tPA-based thrombolytic stroke therapy.

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Figures

Figure 1

Figure 1

Effect of rA2 on tPA-dependent plasmin generation in vitro. (A) Plasmin activity was measured as described under Materials and methods and expressed as a ratio to that generated by either 2.5 _μ_g/mL of tPA alone or 100 Units/mL of uPA alone. Concentrations of both rA2 and BSA were 5 _μ_g/mL. Data were expressed as mean+s.e.m., *P<0.001, _n_=4 per group. (B) A range of concentrations of tPA (1, 2.5, 5, 10 _μ_g/mL) with or without the indicated concentrations of rA2 (0, 1, 2.5, 5 _μ_g/mL) were added to wells of 96-well plate. Plasmin activity was represented as fold of plasmin activity related to 1 _μ_g/mL of tPA alone. Data were expressed as mean+s.e.m., _n_=4 per group.

Figure 2

Figure 2

Effect of treating rats at 2 h after initiation of ischemia. (A) Two hours after initiation of ischemia, animals were treated intravenously with saline, high-dose tPA (10 mg/kg, H-tPA), intermediate-dose tPA (5 mg/kg, M-tPA), low-dose tPA (2.5 mg/kg, L-tPA), rA2 alone (5 mg/kg), or a combination of low-dose tPA (2.5 mg/kg) plus rA2 (5 mg/kg). Laser-doppler flowmetry was used to monitor regional cerebral blood flow (rCBF) for up to 1 h after treatment. (B) At 24 h after stroke, brain infarction was stained by TTC, and the volume was quantified using computer-assisted image analysis. Data were expressed as mean+s.e.m., *P<0.05 for L-tPA plus rAN, #P<0.05 for H-tPA, respectively, _n_=7 or 8 per group.

Figure 3

Figure 3

Effect of treating rats at 4 h after initiation of ischemia. (A) Representative images of brain sections after TTC staining at 24 h after initiating ischemia. At 4 h after stroke onset, three groups of rats were treated intravenously with saline, high-dose tPA (10 mg/kg, H-tPA), or low-dose tPA (2.5 mg/kg, L-tPA) plus rA2 (5 mg/kg). Ischemic infarctions (white color area) were detected in all three groups; however, large areas of grossly visible hemorrhage appeared only on the brain sections of H-tPA-treated rats pointed by arrows. (B) At 24 h after stroke, brain infarction was quantified using computer-assisted image analysis. (C) Volumes of intracerebral hemorrhage ware quantified with hemoglobin assay at 24 h after stroke. Data were expressed as mean+s.e.m., *P<0.05, _n_=13 for saline, _n_=10 for H-tPA, _n_=14 for the combination.

Figure 4

Figure 4

Effect of tPA alone or in combination with rA2 on plasma levels of -dimer. Plasma samples were collected before ischemia, just before thrombolytic therapy, and 1 h after treatment. Concentrations of -dimer in plasma were quantified by ELISA analysis. Data were expressed as mean+s.e.m., *P<0.01 versus ischemia, #P<0.01, _n_=6 per group.

Figure 5

Figure 5

MMP activation in ischemic brains from rats treated 4 h after stroke onset. At 24 h after stroke onset, we performed in situ zymography to examine MMP activation in ischemic brains at delayed 4 h treatments. In the cortex of the periinfarction zone, brains from animals treated with tPA alone showed brighter activated MMP signals compared with saline treatment, but the combination of low-dose tPA plus rA2 had similar or even slightly less positive signals compared with tPA alone treatment. Similar observations were obtained from three individual experiments.

Figure 6

Figure 6

Three days neurological outcomes in rats treated at 4 h after stroke onset. (A) Two groups of rats were treated intravenously at 4 h after stroke with either saline, or a combination of low-dose tPA (2.5 mg/kg, L-tPA) plus rA2 (5 mg/kg). Three days after stroke, neurological scores were significantly improved in treated rats. (B) Ischemic infarctions on H&E-stained sections were quantified using computer-assisted image analysis. Infarction volumes were significantly reduced in the treated rats. *P<0.05, _n_=8 for saline, _n_=11 for the combination.

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