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Background and Purpose: A recombinant engineered variant of APC (activated protein C), 3K3A-APC, lacks anticoagulant properties (<10%) while preserving APCs anti-inflammatory, anti-apoptotic, and neuroprotective functions and is very promising in clinical trials for ischemic stroke. Therapeutic intervention with single bolus administration of the third-generation tPA (tissue-type plasminogen activator), tenecteplase, is anticipated to be widely adopted for treatment of acute ischemic stroke. 3K3A-APC is well-tolerated in stroke patients dosed with alteplase, and in vitro studies show 3K3A-APC does not interfere with alteplase-induced clot lysis. The purpose of this in vitro study was to assess the influence of 3K3A-APC on tenecteplase-induced clot lysis., Methods: Tenecteplase-mediated lysis of thrombin generated plasma clots of human normal pooled plasma was monitored in the presence of varying doses of 3K3A-APC. The effects on fibrinolysis by tenecteplase and alteplase were compared., Results: The presence of 3K3A-APC shortened the time for clot lysis induced by tenecteplase at very low levels but not at higher therapeutic concentrations of tenecteplase. Comparisons of alteplase-mediated clot lysis to tenecteplase clot lysis showed that both thrombolytic agents behaved similarly in the presence of 3K3A-APC., Conclusions: These results indicate that 3K3A-APC does not interfere with tenecteplase's clot lysis function.

Background and Purpose: 3K3A-activated protein C (APC) protects young, healthy male rodents after ischemic stroke. 3K3A-APC is currently under development as a neuroprotectant for acute ischemic stroke in humans. Stroke Therapy Academic Industry Roundtable recommends that after initial studies in young, healthy male animals, further studies should be performed in females, aged animals, and animals with comorbid conditions. Here, we studied the effects of delayed 3KA-APC therapy alone and with tissue-type plasminogen activator (tPA) in aged female mice and spontaneously hypertensive rats., Methods: We used Stroke Therapy Academic Industry Roundtable recommendations for ensuring good scientific inquiry. Murine recombinant 3K3A-APC (0.2 mg/kg) alone or with recombinant tPA (10 mg/kg) was given intravenously 4 hours after transient middle cerebral artery occlusion in aged female mice and rats and after embolic stroke in spontaneously hypertensive rat. 3K3A-APC was additionally administered within 3 to 7 days after stroke. The neuropathological analysis and neurological scores, foot-fault, forelimb asymmetry, and adhesive removal tests were performed within 7 and 28 days of stroke., Results: In all models, tPA alone had no effects on the infarct volume or behavior. 3K3A-APC alone or with tPA reduced the infarct volume 7 days after the middle cerebral artery occlusion in aged female mice and embolic stroke in spontaneously hypertensive rat by 62% to 66% and 50% to 53%, respectively, significantly improved (P<0.05) behavior, and eliminated tPA-induced intracerebral microhemorrhages. In aged female mice, 3K3A-APC was protective within 4 weeks of stroke., Conclusions: 3K3A-APC protects from ischemic stroke and extends the therapeutic window of tPA in aged female mice and in spontaneously hypertensive rat with a comorbid condition.

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.

Background and Purpose: Activated protein C (APC), a protease with anticoagulant and cytoprotective activities, protects neurons and endothelium from ischemic injury. Drotrecogin-alfa activated, a hyperanticoagulant form of human recombinant APC, is currently being studied in patients with ischemic stroke. How changes in APC anticoagulant activity influence APC's neuroprotection and risk for bleeding is not clear., Methods: We used neuronal and brain endothelial cell injury models and middle cerebral artery occlusion in mice to compare efficacy and safety of drotrecogin-alfa activated and human 3K3A-APC, an APC nonanticoagulant mutant., Results: Drotrecogin-alfa activated and 3K3A-APC exhibited 148% and 10% of plasma-derived APC's anticoagulant activity and differ in the carbohydrate content. 3K3A-APC protected mouse neurons from N-methyl-d-aspartate-induced apoptosis and human brain endothelial cell from oxygen-glucose deprivation with 1.8- and 3.1-fold greater efficacy than drotrecogin-alfa activated. Given 5 minutes before transient middle cerebral artery occlusion, 3K3A-APC and drotrecogin-alfa activated (0.5 and 2 mg/kg intravenously) reduced comparably and dose-dependently the infarction lesion up to 85%. 3K3A-APC, but not drotrecogin-alfa activated, improved neurological score dose-dependently (P<0.05). 3K3A-APC did not cause bleeding. In contrast, drotrecogin-alfa activated dose-dependently increased hemoglobin content in postischemic brain. After permanent middle cerebral artery occlusion, 3K3A-APC multidose therapy (1 mg/kg intravenously at 12 hours and 1, 3, 5, and 7 days) improved functional recovery and reduced infarction by 60% with no risk for bleeding, whereas drotrecogin-alfa activated increased hemoglobin deposition in the postischemic brain and showed relatively modest neuroprotection., Conclusions: Nonanticoagulant 3K3A-APC exhibits greater neuroprotective efficacy with no risk for bleeding compared with drotrecogin-alfa activated, a hyperanticoagulant form of APC.

Accumulation of amyloid beta-peptide (Abeta) in the central nervous system (CNS) may initiate pathogenic cascades mediating neurovascular and neuronal dysfunctions associated with the development of cerebral beta-amyloidosis and cognitive decline in patients with Alzheimer disease (AD) and with related familial cerebrovascular disorders. Whether Abeta-related pathology in the CNS is reversible or not and what key therapeutic targets are controlling Abeta/amyloid levels in the aging brain remain debatable. In this article, we summarize recent evidence why the receptor for advanced glycation end products and low-density lipoprotein receptor related protein 1 in the vascular CNS barriers are critical for regulation of Abeta homeostasis in the CNS and how altered activities in these 2 receptors at the blood-brain barrier may contribute to the CNS Abeta accumulation resulting in neuroinflammation, disconnect between the cerebral blood flow and metabolism, altered synaptic transmission, neuronal injury, and amyloid deposition into parenchymal and neurovascular lesions. We briefly discuss the potential of advanced glycation end products and low-density lipoprotein receptor related protein 1-based therapeutic strategies to control brain Abeta in animal models of AD and ultimately in patients with AD and related familial cerebrovascular beta-amyloidoses.

Background and Purpose: Tissue plasminogen activator (TPA) is normally expressed in rat brain capillaries. This study examines the expression of TPA in brain capillaries of diabetic rats in relation to focal ischemic brain injury., Methods: Diabetes type 1 was induced by streptozotocin for 7 days. Acute hyperglycemia was induced by 50% dextrose. Expression of TPA in brain capillaries was determined by Western blot and reverse transcription-polymerase chain reaction analyses. Focal stroke was produced by 1 hour of reversible middle cerebral artery occlusion. Physiological variables and cerebral blood flow were monitored during occlusion and within 1 hour of reperfusion. Neurological and neuropathologic examinations were performed after 24 hours of reperfusion., Results: All rats developed comparable hyperglycemia (approximately 15 mmol/L). A complete depletion of TPA protein and 6.5-fold decrease in TPA mRNA were found in brain capillaries of diabetic rats, in contrast to normal TPA capillary levels in hyperglycemic rats. The blood flow in the periphery of the ischemic core was significantly reduced during reperfusion by 52% to 62% (P<.001) in diabetic rats and by 23% to 25% (P<.05) in hyperglycemic rats. The neurological score was worsened by 3.2-fold (P<.0003) by diabetes and by 24% by hyperglycemia only. Significant 41% (P<.007) and 29% (P<.05) increases in infarct volume and 163% (P<.007) and 60% increases in edema volume were found in diabetic rats relative to control and hyperglycemic rats, respectively., Conclusions: Diabetes type 1, but not acute hyperglycemia, produces downregulation of TPA in rat brain capillaries. This TPA reduction is associated with impaired restoration of blood flow after an ischemic insult, poor neurological outcome, and enhanced ischemic brain injury.