Alteplase

At 60 minutes post-occlusion, animals were intravenously administered tPA (Alteplase, Boehringer Ingelheim, Australia) at the clinical dose (0.9 mg/kg, n = 6), 2x the clinical dose (1.8 mg/kg, n = 6), 5x the clinical dose (4.5 mg/kg, n = 6), or the rat dose (10 mg/kg, n = 7) as a 10% bolus over 1 minute, with the remainder infused over 1 hour.
The 0.9 mg/kg and 10 mg/kg groups were part of a separate study in which they had been randomised to either tPA or tPA+ultrasound treatment (tPA + ultrasound groups not presented here). The additional animals were randomized to either 1.8 or 4.5 mg/kg tPA for this study.

Dosage of 3 mg/kg (1 mg/ml) rt-PA (Alteplase, Boehringer Ingelheim AB, Germany) was chosen after evaluation of 1, 3, and 10 mg/kg, where 1 mg/kg showed no improvement and 10 mg/kg caused bleedings (data not shown). Treatment (rt-PA or saline) was randomized and administrated intravenously, starting with a 10% bolus dose followed by a 40 min infusion administrated either at 1 h or 4 h after stroke induction. Animals treated at the late time point were awakened 1 h after stroke induction and then re-anaesthetized for treatment at 4 h.

Patients in the two groups were treated according to the treatment regimen of ACI described in the 2014 Guidelines for Diagnosis and Treatment of Acute Ischemic Stroke in China (17 ), including electrocardiogram monitoring of vital signs, oxygen inhalation, monitoring and controlling blood pressure and blood glucose, dehydration for releasing intracranial pressure, antiplatelet and anticoagulation therapy, the use of free-radical scavenger such as edaravone and other neuroprotective agents, and others. After vital signs were stable, the patients were guided to perform functional rehabilitation.
Patients in the observation group were given alteplase (Boehringer Ingelheim, Germany) at a total dosage of 0.9 mg/kg, in addition to general treatment. Ten percent of the total dosage was injected intravenously within 1 min, and the remaining 90% was dripped intravenously over 1 h. The course of treatment lasted 14 days.
Patients in the control group were given batroxobin (Beijing Tobishi Pharmaceutical Co., Ltd., China), in addition to general treatment. The initial dose on the first day of admission was 10 batroxobin units (BU), which were dripped intravenously over 1 h, and on the third day, 5 BU batroxobin was dripped intravenously over 1 h. The course of treatment lasted 14 days.

Patients were enrolled between March 2011 and January 2013 in a single Stroke Center (Department of Neurology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary). Study population included consecutive acute ischemic stroke patients admitted within 4.5 hours of their symptom onset undergoing intravenous (i.v.) thrombolytic therapy according to the European Stroke Organization guidelines [6 (link)] using recombinant tissue plasminogen activator (rt-PA, Alteplase, Boehringer Ingelheim, Germany). Patients who were on anticoagulant therapy at admission were excluded from the study population. All enrolled patients or their relatives had been informed about the study and gave written informed consent. The study was approved by the Ethics Committee of University of Debrecen, Debrecen, Hungary.

Mice that underwent MCAO surgery were randomised into three treatment groups. Mice were injected intravenously (i.v.) with either: (1) vehicle only (200 μL of 2% HSA in saline) after 30 min of reperfusion, (2) tPA (Alteplase, 10 mg/kg; Boehringer Ingelheim) at the start of reperfusion and then vehicle after 30 min, or (3) tPA at the start of reperfusion and then hAECs (1×10⁶ cells in 200 μL of 2% HSA in saline) after 30 min. A further 15 mice were sham-operated and then treated with vehicle (n = 7) or tPA + vehicle (n = 8) after 30 min. For sham surgery, we made a neck incision and exposed the CCA, ECA, ICA and bifurcation. No further surgical procedure or vessel ligation was made. Mice were euthanised according to designated endpoint times at 3 h (n = 21), 6 h (n = 31) or 24 h (n = 50) post-stroke. Animals euthanised at 24 h were assessed for functional deficits in two tests (see below). Brains were collected to determine infarct volume and assess markers of injury and inflammation.

The effect of M3P during clot formation and lysis was studied by monitoring the change in turbidity in human or mouse plasma using a microplate reader (FLUOstar Optima, BMG Labtech). Calcium chloride (final concentration, 25 mM) was added to citrated plasma diluted 1:2 in Hepes buffer [10 mM Hepes, 150 mM NaCl, and 0.4% bovine serum albumin (BSA) (pH 7.4)] to promote coagulation. Samples were incubated at 37°C with M3P at different doses, and absorbance (405 nm) was monitored for 12 hours every 30 s at 37°C. When appropriate, recombinant tissue-type plasminogen activator (alteplase, Boehringer Ingelheim) was also added to promote clot lysis. Results are expressed as the time to achieve 75% maximal absorbance [clotting time (CT)], and the 50% lysis time was calculated as the time from initiation of clot formation to the time at which maximal absorbance falls to 50%. All experiments were performed in triplicate.