An activated protein C analog with reduced anticoagulant activity extends the therapeutic window of tissue plasminogen activator for ischemic stroke in rodents

Abstract

Background and purpose: Tissue plasminogen activator (tPA) is the only approved therapy for acute ischemic stroke. However, tPA has a brief therapeutic window. Its side effects include intracerebral bleeding and neurotoxicity. Therefore, a combination therapy with tPA and agents that can extend the therapeutic window of tPA and/or counteract its side effects are warranted. Here, we studied whether 3K3A-APC, a neuroprotective analog of activated protein C with reduced anticoagulant activity, can enhance the therapeutic effects of tPA in models of ischemic stroke in rodents.

Methods: Human recombinant tPA (10 mg/kg), alone or in combination with human recombinant 3K3A-APC (2 mg/kg), was administered intravenously 4 hours after proximal or distal transient middle cerebral artery occlusion in mice and embolic stroke in rats. The 3K3A-APC was additionally administered for 3 to 4 consecutive days after stroke. The neuropathological and neurological analyses were performed at 1 to 7 days after stroke.

Results: In all models, tPA alone had no effects on the infarct volume or behavior (ie, neurological score, foot-fault, forelimb asymmetry, adhesive removal) compared with vehicle. The tPA and 3K3A-APC combination therapy reduced the infarct volume 24 hours and 7 days after proximal or distal transient middle cerebral artery occlusion in mice and 7 days after embolic stroke in rats by 65%, 63%, and 52%, respectively, significantly (P<0.05) improved behavior and eliminated tPA-induced intracerebral microhemorrhages.

Conclusions: The 3K3A-APC extends the therapeutic window of tPA for ischemic stroke in rodents. Therefore, this combination therapy also should be considered for treating stroke in humans.

Figure 1. Human recombinant 3K3A-APC enhances the therapeutic effects of tPA after proximal transient MCAo in mice

tPA (10 mg/kg) and human 3K3A-APC (2 mg/kg) were administered intravenously 4 hours after 45 min proximal transient MCAo. (a–d) Infarct volume (a), edema (b), motor neurological score (c) and hemoglobin levels in the ischemic hemipshere (d) were determined 24 hours after the MCAo. All values are mean ± SEM, n = 5 mice per group.

Figure 2. Human recombinant 3K3A-APC enhances the therapeutic effects of tPA after distal transient MCAo in mice

tPA (10 mg/kg) and human 3K3A-APC (2 mg/kg) were administered intravenously 4 hours after 60 min distal transient MCAo. The additional doses of human 3K3A-APC (2 mg/kg, intraperitoneally) were administered at 1, 3, 5, and 7 days after the MCAo. (a–b) Foot-fault test (a) and forelimb asymmetry test (b) were performed at day 1, 3 and 7 after stroke. (c–d) The infarct volume (c) and hemoglobin levels in the ischemic hemisphere (d) were determined 7 days after the MCAo. All values are mean ± SEM, n = 5 mice per group. ^aP < 0.01, vehicle versus tPA + 3K3A-APC; ^bP < 0.01, tPA alone versus tPA + 3K3A-APC.

Figure 3. Effects of combined tPA and 3K3A-APC treatment on behavior in rats after embolic stroke

tPA (10 mg/kg) and human 3K3A-APC (2 mg/kg) were administered intravenously 4 hours after embolic stroke. 3K3A-APC (2 mg/kg) was additionally injected intravenously for 3 consecutive days. (a–c) Modified neurological severity score (mNSS) (a), foot-fault test (b) and adhesive removal test (c) were performed at day 1 and 7 after stroke. (e) Microscopic hemorrhage was determined 7 days after stroke. All values are mean ± SEM, n = 10 rats per group.

Figure 4. Effects of combined tPA and 3K3A-APC treatment on the infarct volume and microscopic hemorrhage in rats after embolic stroke

Rats that were studied for behavior in Figure 3 were sacrificed after 7 days of stroke to determine (a) the infarct volume and (b) microscopic hemorrhage. All values are mean ± SEM, n = 10 rats per group.