Tenecteplase in Wake-up Ischaemic Stroke Trial (TWIST). A Randomised-controlled Trial of Thrombolytic Treatment With Tenecteplase for Acute Ischaemic Stroke Upon Awakening
Phase: PHASE3
Conditions: Ischemic Stroke, Stroke, Acute
Sponsor: University Hospital of North Norway

 
 
 
 
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Protocol 
 
Tenecteplase in Wake-up Ischaemic Stroke Trial (TWIST) 
 
 
A randomised-controlled trial of thrombolytic treatment with tenecteplase for acute ischaemic 
stroke upon awakening 
 
EudraCT number: 2014-000096-80 
Sponsor’s protocol code number: 01 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sponsor: 
University Hospital of North Norway, NO-9038 Tromsø, Norway 
Telephone number: +47 77626000 
 
 
 
Trial Coordinating Centre: 
Brain and Circulation Research Group, Department of Neurology, University Hospital of North 
Norway, and Faculty of Health Sciences, University of Tromsø 
Address: Department of Neurology, University Hospital of North Norway, NO-9038 Tromsø, 
Norway 
Telephone number: +47 77627120 
 
 

 
 
 
 
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Signature page 
 
 
 
 
 
Sponsor 
 
 
 
_________________________________________________________________ 
Director of Research (signature and date) 
University Hospital of North Norway 
 
 
 
 
_________________________________________________________________ 
Trial Coordinating Investigator and Head of Brain and Circulation Research Group  
(signature and date) 
University Hospital of North Norway and University of Tromsø 
 
 
 
 
 
Principal investigator 
 
I have read this protocol and agree that it contains all the necessary details for carrying out the 
study. I will conduct the study as outlined herein and will complete the study within the time 
designated. 
 
I will provide copies of the protocol and all pertinent information to all individuals responsible to 
me who assist in the conduct of the study. I will discuss this material with them to ensure that they 
are fully informed regarding the study intervention and the conduct of the study. 
 
 
 
_________________________________________________________________ 
Principal investigator (signature and date) 
 
 
 
 
_________________________________________________________________ 
Name of institution 
 
 
 
 
 
 

 
 
 
 
Protocol version/date 200917
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1. Contents 
1. 
Contents.............................................................................................................................. 3 
2. 
Summary ............................................................................................................................ 5 
3. 
Background and rationale................................................................................................... 5 
3.1 
Introduction ...................................................................................................................... 5 
3.2 
Potential benefits and harms of tenecteplase .................................................................... 5 
3.3 
Multi-modal CT for the selection or patients to thrombolytic therapy ............................. 7 
4. 
Trial design, questions, and hypotheses ............................................................................. 8 
4.1 
Trial design ....................................................................................................................... 8 
4.2 
Study questions and objectives ......................................................................................... 8 
4.3 
Hypotheses ....................................................................................................................... 8 
5. 
Study population ................................................................................................................ 8 
5.1 
Inclusion criteria ............................................................................................................... 8 
5.2 
Exclusion criteria .............................................................................................................. 9 
6. 
Patient screening ................................................................................................................ 9 
6.1 
Patient screening ............................................................................................................... 9 
6.2 
Non-contrast CT and CT angiography for patient screening before randomisation ....... 10 
6.3 
CT perfusion at selected centres before randomisation (substudy) ................................ 10 
6.4 
Routine use of MRI before randomisation ..................................................................... 10 
7. 
Patient inclusion ............................................................................................................... 10 
7.1 
Patient information and consent/assent .......................................................................... 10 
7.2 
Randomisation procedure and recording of baseline characteristics .............................. 10 
8. 
Trial treatment .................................................................................................................. 11 
8.1 
Open-label tenecteplase plus best standard care ......................................................... 11 
8.2 
Best standard care alone ............................................................................................. 11 
8.3 
Best standard care and other treatments ..................................................................... 11 
8.4 
Management of serious adverse events related to study treatment ............................. 11 
9. 
Visits and examinations from trial entry till end of follow-up ......................................... 12 
9.1 
Visits and examinations during stay in hospital ......................................................... 12 
9.2 
Centralised follow-up via telephone or mail at 3 months (90 ±7 days) ...................... 12 
9.3 
Long-term follow-up by record linkage with central registries ................................ 122 
10. Effect variables ................................................................................................................. 13 
10.1 
Primary effect variable ................................................................................................... 13 
10.2 
Secondary effect variables .............................................................................................. 13 
11. Analysis and statistical considerations ............................................................................. 13 
11.1 
Estimation of sample size ............................................................................................... 13 
11.2 
Statistical analyses .......................................................................................................... 14 
12. Trial conduct and practices/procedures ............................................................................ 15 
12.1 
Compliance with regulations and guidelines .................................................................. 15 
12.2 
Data protection ............................................................................................................... 15 
12.3 
Ethical conduct ............................................................................................................... 15 
12.4 
Liability .......................................................................................................................... 15 
12.5 
Recording and reporting of serious adverse events ...................................................... 155 
12.6 
Event adjudication .......................................................................................................... 16 
12.7 
Monitoring of data quality .............................................................................................. 16 
12.8 
Handling of protocol violations and protocol amendments ............................................ 16 
12.9 
Monitoring of effectiveness and safety .......................................................................... 16 
12.10 Audit and inspection ....................................................................................................... 17 
12.11 Handling of patient data ................................................................................................. 17 
12.12 Handling of the list of treatment codes ........................................................................... 17 
12.13 Screening logs ................................................................................................................ 17 
12.14 Financial conduct ............................................................................................................ 17 
12.15 Publication and data sharing policy ................................................................................ 17 
12.16 User involvement ............................................................................................................ 17 
13. Central trial organisation .................................................................................................. 17 
13.1 
Sponsors and funding bodies .......................................................................................... 17 

 
 
 
 
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13.2 
Trial Coordinating Centre ............................................................................................... 18 
13.3 
Trial Statistical Centre .................................................................................................... 18  
13.4      Executive Committee...................................................................................................... 18 
13.5 
Imaging Scientific Committee ........................................................................................ 18 
13.6 
Trial Steering Committee ............................................................................................... 18 
13.7 
Data Monitoring Committee ........................................................................................... 18 
13.8 
Event Adjudication Committee ...................................................................................... 18 
14. Time table and end of trial ............................................................................................... 18 
15. References ........................................................................................................................ 19 
16. Appendix .......................................................................................................................... 23 
16.1 
On-going studies of thrombolytic treatment for wake-up stroke .................................... 23 
16.2 
Definitions of clinical events .......................................................................................... 23 
16.2.1 
Serious adverse events ............................................................................................ 23 
16.2.2 
Death ...................................................................................................................... 23 
16.2.3 
Recurrent stroke  ..................................................................................................... 24 
16.2.4 
Symptomatic intracranial haemorrhage  ................................................................. 24 
16.2.5 
Asymptomatic intracranial haemorrhage ................................................................ 24 
16.2.6 
Neurological deterioration due to index stroke  ...................................................... 23 
16.2.7 
Symptomatic intracranial haemorrhage  ................................................................. 24 
16.2.8 
Asymptomatic intracranial haemorrhage ................................................................ 24 
16.3 
Abbreviations ................................................................................................................. 25 
17. Summary of changes ........................................................................................................ 25 
17.1 
Protocol version/date 160323 ......................................................................................... 25 
17.2 
Protocol version/date 160421 ......................................................................................... 26 
17.3 
Protocol version/date 170412 ......................................................................................... 26 
17.4 
Protocol version/date 180410 ......................................................................................... 26 
17.5 
Protocol version/date 180704 ......................................................................................... 26 
17.6 
Protocol version/date 200131 ......................................................................................... 26 
17.7 
Protocol version/date 200917 ......................................................................................... 26 
 
 
 
 

 
 
 
 
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2. Summary 
Tenecteplase in Wake-up Ischaemic Stroke Trial (TWIST) is a randomised-controlled, open-label 
trial of thrombolytic treatment with tenecteplase for acute ischaemic stroke upon awakening. 
 
The trial aims to answer the following questions:  
 
Can thrombolytic treatment with tenecteplase given within 4.5 hours of wake-up reduce the 
risk of poor functional outcome at 3 months? 
 
Can findings on non-contrast CT and CT angiography (and CT perfusion at selected centres) 
identify patients who benefit from such treatment, compared to patients without such 
findings? 
 
Patients are eligible if they have ischaemic stroke causing measurable neurologic deficits, and can 
be given tenecteplase within 4.5 hours after awakening. Patients will undergo brain CT and CT 
angiography (if possible) to exclude large infarction or other contraindications to thrombolytic 
treatment. Non-contrast CT and CT angiography (if possible) will be repeated on day 2. 
 
Patients will be allocated to tenecteplase 0.25 mg/kg as a bolus (maximum dose 25 mg) or to 
control. The primary effect variable is functional outcome at 3 months, as measured by the 
modified Rankin Scale (mRS). 
 
The target is to include a total of 600 patients from centres in Norway, Sweden, Denmark, 
Finland, Estonia, Lithuania, Latvia, Switzerland, New Zealand and the United Kingdom. The 
sponsor of the trial is University Hospital of North Norway. 
 
3. Background and rationale 
3.1 Introduction 
Thrombolytic treatment with recombinant tissue plasminogen activator (rt-PA) given within 4.5 
hours of onset improves clinical outcome after ischaemic stroke, despite an increased risk of 
intracranial haemorrhage.1,2 Patients who have new stroke symptoms when they wake up from 
sleep (“wake-up stroke”) are excluded for such treatment, because the time of stroke onset is 
unknown. About one in five strokes occur during sleep,3-5 and it is unknown whether patients with 
wake-up stroke will benefit from thrombolytic treatment. 
 
There are reasons to expect that patients with wake-up stroke will benefit from treatment. Several 
studies have shown that the onset of stroke during sleep is close to awakening,6,7 and that patients 
with wake-up stroke share many clinical and radiological findings with patients with stroke 
duration less than 4.5 hours.8-13 Studies have also shown that thrombolytic treatment is safe in 
patients with wake-up stroke up to 4.5 hours from wakening,14-19 and recently the WAKE-UP trial 
showed benefit of treatment with alteplase in patients selected based on DWI/FLAIR mismatch on 
MRI.20 In a Cochrane systematic review21 we have identified only one completed, small 
randomised-controlled trial of only nine patients.22 Two trials have recently been completed 
(EXTEND,23  THAWS,24) and one is on-going (WASSABI,25  see Appendix). All these three 
trials use alteplase, and all use advanced imaging techniques for selection of patients. We will use 
a different strategy, and perform a large trial of tenecteplase, using a pragmatic study design, 
without exclusion of patients based on MRI findings. The trial will be complementary to the other 
trials, and is planned in advance to be included in our Cochrane meta-analysis of all trials of 
thrombolytic treatment for wake-up stroke, so that smaller, but still worthwhile effects can be 
detected that may be missed by the individual trials. 
 
3.2 Potential benefits and harms of tenecteplase 
 
Pharmacological properties 

 
 
 
 
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Tenecteplase is an alteplase molecule that is genetically engineered to have pharmacological 
advantages over alteplase: it has a 14-fold greater fibrin specificity and a very rapid onset of 
action compared to alteplase, has a longer half-life, and can be given as a single bolus, whereas 
alteplase must be given as a continuous infusion over one hour.26,27 Tenecteplase also has a 
smaller effect on plasma fibrinogen levels,28 which is beneficial for avoidance of intracranial 
haemorrhage,29 and weaker pro-thrombotic effects.30-32  
 
Data from pre-clinical studies 
Animal models have shown that tenecteplase acts faster33 and more potent33-35 than alteplase, and 
that tenecteplase produces a more rapid and complete recanalisation of occluded arteries.31,34,36 
Models have also shown that experimentally induced infarcts were less frequently converted to 
haemorrhage with tenecteplase than with bio-equivalent doses of alteplase36,37 and that the risk 
peripheral or major haemorrhage31 and intracranial haemorrhage36-41 was equal to or lower than 
the risk with alteplase. 
 
Data from clinical studies 
Tenecteplase is preferred over alteplase for treatment of myocardial infarction, because it has 
lower risk of bleeding complications, and because of the pharmacological advantages listed 
above.42 
 
A number of observational studies in patients with ischaemic stroke have shown that, compared to 
alteplase, tenecteplase is associated with similar or better recanalisation rates and clinical 
outcomes, and a similar or lower risk of intracranial haemorrhage.43-47 These studies have also 
concluded that the optimal dose of tenecteplase for stroke is 0.25 mg/kg.28,45,48,49 
 
There has also been three small randomised-controlled trials of tenecteplase vs. alteplase in acute 
ischaemic stroke.45,47,48 Meta-analysis of these trials with a total of 283 patients have shown that 
76/178 patients (43%) patients given tenecteplase (doses ranging from 0.1 to 0.4 mg/kg) achieved 
good functional outcome (mRS score 0-1), compared to 33/105 patients (31%) patients given 
alteplase (odds ratio (OR) 1.39, 95% confidence interval (CI) 0.82-2.36, Figure 1). 
 
Figure 1. Good functional outcome (mRS score 0-1) at three months 
 
 
There was also a non-significant lower risk of symptomatic intracranial haemorrhage in patients 
given tenecteplase (Figure 2). The risks were 7/131 (5%) in patients given tenecteplase and 4/56 
(7%) in patients given alteplase (OR 0.71, 95% CI 0.11-4.42). ATTEST 2014 did not provide data 
on symptomatic intracranial haemorrhage. 
 
Figure 2. Symptomatic intracranial haemorrhage 
 
 
There was no significant difference in the risk of death at three months (Figure 3). In total, 24 of 
the 178 patients (13%) given tenecteplase and 17 of the 123 patients (14%) given alteplase were 
dead at three months (OR 1.00, 95% CI 0.52-1.95). 

 
 
 
 
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Figure 3. Death of all causes at three months 
 
 
Another meta-analysis comparing different doses of tenecteplase concluded that 0.25 mg/kg is the 
preferred dose over alteplase.50 There are also observational studies that support this 
conclusion.28,49 Furthermore, an analysis of pooled data for tenecteplase 0.25 mg/kg compared to 
alteplase 0.9 mg/kg showed trends in favour of tenecteplase.50 Tenecteplase had higher odds than 
alteplase for neurological improvement at 24 hours (OR 3.3, 95% CI 1.5-7.2, p=0.09), excellent 
clinical outcomes at three months (OR 1.9, 95% CI 0.8-4.4, p=0.3), and good clinical outcomes at 
three months (OR 1.9, 95% CI 0.5-7.2, p=0.4); also, tenecteplase had a lower risk of brain 
haemorrhage than alteplase (OR 0.3, 95% CI 0.2-1.8, p=0.4). Recently, NOR-TEST showed no 
difference in functional outcome at three months between patients with ischaemic stroke treated 
with tenecteplase 0.4 mg/kg or alteplase 0.9 mg/kg within 4.5 hours51 and the EXTEND-IA TNK 
trial showed that tenecteplase 0.25 mg/kg was superior to alteplase 0.9 mg/kg in improving 
recanalisation and functional outcome at three months when given within 4.5 hours and prior to 
intra-arterial intervention.52 Other trials of tenecteplase in patients with acute ischaemic stroke are 
ongoing: TASTE53, TEMPO-254 and ATTEST2.55 
 
Conclusion: Potential benefits and harms of tenecteplase  
The bolus administration and the very rapid onset of action makes tenecteplase an attractive 
option for patients with wake-up stroke, because the time to recanalisation of an occluded cerebral 
artery can be reduced by up to one hour, compared to alteplase.17 Available data from 
pharmacological in-vitro studies, pre-clinical studies and clinical studies also indicate that 
tenecteplase 0.25 mg/kg is at least as safe and at least as effective as alteplase for the treatment of 
acute ischaemic stroke. The favourable profile of tenecteplase seen in these studies and in studies 
of acute myocardial infarction, in combination with the pharmacological characteristics and 
advantages of bolus administration, provides a good rationale for testing tenecteplase in patients 
with wake-up stroke. 
 
3.3 Multi-modal CT for the selection of patients to thrombolytic therapy 
The WAKE-UP trial and three other trials of thrombolytic treatment for wake-up stroke use/used 
advanced imaging techniques for selection of patients.20,23-25 Two trials (WASSABI25 and 
EXTEND23) use CT or MRI perfusion techniques to identify patients with ischaemic penumbra. 
The penumbra is an area of ischaemic, but still viable brain tissue surrounding the core of a 
cerebral infarction, which is assumed to be salvageable if blood flow can be promptly restored. 
The WAKE-UP trial20 and the THAWS24 used DWI/FLAIR mismatch  on MRI to select patients. 
Infarct changes on DWI are thought to represent alterations to water diffusion and can be 
visualised within minutes of onset. Infarct changes on FLAIR are thought to represent cytotoxic 
oedema and cannot be identified until a few hours after onset. Findings suggestive of ischaemic 
stroke on DWI, but absence of such findings on FLAIR (DWI/FLAIR mismatch), may therefore 
identify patients with a short time since onset of cerebral infarction. 
 
Demonstration of penumbra or DWI/FLAIR mismatch identifies patients with a good prognosis, 
but it does not necessarily identify patients with a better response to thrombolytic treatment than 
other patients. Only one randomised-controlled trial (MR RESCUE, of intra-arterial interventions) 
has examined the value of advanced imaging for patient selection56, but did not find a better effect 
of therapy in patients with penumbra, than in patients without penumbra.56 

 
 
 
 
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On the contrary, there is a risk that selection of patients based on such techniques will exclude 
patients from receiving an effective treatment. First, it is possible that patients without these 
specific radiological findings benefit equally well as those who are selected for participation in 
these trials, as indicated in a recent study.19 Second, the techniques are far from perfect in 
characterising patients, for example, DWI/FLAIR mismatch can be absent in as many as 40% of 
patients with known stroke duration of less than three hours.57 Third, these MRI techniques are 
time-consuming and are not available in the emergency setting in many hospitals, which means 
that time to thrombolytic treatment will be increased, and that some patients will be denied 
thrombolytic treatment altogether. 
 
We will therefore base patient inclusion on wider and more pragmatic criteria, such as time since 
wake-up in combination with accepted CT exclusion criteria. Inclusion based on wider criteria 
will avoid excluding a large number of patients that might benefit from treatment. This will make 
the results applicable to patients that are encountered in everyday clinical practice, and will 
dramatically increase the proportion of stroke patients who can be treated. Patient can be included 
based on non-contrast CT alone, but CT angiography can be routinely done in many hospitals, can 
be done without significant delay, and can give information that will help in the selection of 
patients with wake-up stroke for thrombolytic treatment.58-62 
 
4. Trial design, questions, and hypotheses 
4.1 Trial design 
Randomised-controlled, open-label trial of tenecteplase in patients with acute ischaemic stroke 
upon awakening.  
 
4.2 Study questions and objectives 
The objectives of the trial is to answer the following questions, for patients who have an acute 
ischaemic stroke upon awakening: 
 
Can thrombolytic treatment with tenecteplase given within 4.5 hours of wake-up reduce the 
risk of poor functional outcome at 3 months? 
 
Can findings on non-contrast CT and CT angiography (and CT perfusion at selected centres) 
identify patients who benefit from such treatment, compared to patients without such 
findings? 
 
4.3 Hypotheses 
 
Thrombolytic treatment with tenecteplase will reduce the risk of poor functional outcome at 3 
months. For details about size of effect, see Statistical calculations, below. 
 
CT angiography and CT perfusion identifies patients who benefits more from tenecteplase 
than other patients  
 
5. Study population 
Patients with acute ischaemic stroke upon awakening who can be given treatment within 4.5 hours 
after awakening. 
5.1 Inclusion criteria 
 
Stroke symptoms on awakening that were not present before sleep 
 
Clinical diagnosis of stroke with limb weakness with NIHSS score ≥3, or dysphasia 
 
Treatment with tenecteplase is possible within 4.5 hours of awakening 
 
Written consent from the patient, non-written consent from the patient (witnessed by non-
participating health care personnel), or written consent from the nearest family member 

 
 
 
 
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5.2 Exclusion criteria 
 
Age <18 years 
 
NIHSS score >25 or NIHSS consciousness score >2, or seizures during stroke onset 
 
Findings on non-contrast CT that indicate that the patient is unlikely to benefit from 
treatment: 
- Infarction comprising more than >1/3 of the middle cerebral artery territory on non-
contrast CT or CT perfusion 
- Intracranial haemorrhage, structural brain lesions which can mimic stroke (e.g 
cerebral tumour) 
 
Active internal bleeding of high risk of bleeding, e.g.: 
- Major surgery, trauma or gastrointestinal or urinary tract haemorrhage within the 
previous 21 days, or arterial puncture at a non-compressible site within the previous 7 
days 
- Any known defect in coagulation, e.g. current use of vitamin K antagonist with an 
INR >1.7 or prothrombin time >15 seconds, or use of direct thrombin inhibitors or 
direct factor Xa inhibitors during the last 24 hours (unless reversal of effect can be 
achieved by agents such as idarusizumab) or with elevated sensitive laboratory tests 
(such as aPTT, INR, platelet count, eucarin clotting time, TT, or appropriate factor Xa 
activity assays), or heparins during the last 24 hours or with an elevated aPTT greater 
than the upper limit of normal 
- Known defect of clotting or platelet function or platelet count below 100,000/mm3   
(but patients on antiplatelet agents can be included) 
- Ischaemic stroke or myocardial infarction in previous 3 months, previous intracranial 
haemorrhage, severe traumatic brain injury or intracranial or intraspinal operation in 
previous 3 months, or known intracranial neoplasm, arteriovenous malformation or 
aneurysm 
 
Contraindications to tenecteplase, e.g., acute bacterial endocarditis or pericarditis; acute 
pancreatitis; severe hepatic dysfunction, including hepatic failure, cirrhosis, portal 
hypertension; active hepatitis; systemic cancer with increased bleeding risk; haemostatic 
defect including secondary to severe hepatic, renal disease; organ biopsy; prolonged 
cardiopulmonary resuscitation > 2 min (within 2 weeks) 
 
Persistent blood pressure elevation (systolic ≥185 mmHg or diastolic ≥110 mmHg), despite 
blood pressure lowering treatment 
 
Blood glucose <2.7 or >20.0 mmol/L (use of finger-stick measurement devices is acceptable) 
 
Pregnancy, positive pregnancy test, childbirth during last 10 days, or breastfeeding. In any 
woman of childbearing potential, a pregnancy test must be performed and the result assessed 
before trial entry 
 
Other serious or life-threatening disease before the stroke: severe mental or physical disability 
(e.g. Mini Mental Status score <20, or mRS score ≥3), or life expectancy less than 12 months 
 
Patient unavailability for follow-up (e.g. no fixed address) 
 
6. Patient screening 
6.1 Patient screening 
All patients who are admitted with new stroke symptoms on awakening should be screened for 
inclusion into the trial. Patients will be evaluated for vital signs, physical examination, 
neurological status (NIHSS), ECG and CT with CT angiography (section 6.2). Routine blood tests 
will include blood glucose and prothrombin time/INR*, aPTT*, TT and/or eucarin clotting time if 
it is suspected the patient is taking direct thrombin inhibitors or direct factor Xa inhibitors. 
 
*Although it is desirable to know the results of these tests before giving intravenous tenecteplase, fibrinolytic therapy should not be 
delayed while awaiting the results unless (1) there is clinical suspicion of a bleeding abnormality or thrombocytopenia, (2) the patient 
has received heparin or warfarin within 48 hours, or (3) the patient has received other anticoagulants/direct thrombin inhibitors or 
direct factor Xa inhibitors. 
 

 
 
 
 
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6.2 Non-contrast CT and CT angiography for patient screening before 
randomisation 
All patients should be screened with non-contrast CT, and CT angiography should be performed, 
if possible. Findings on non-contrast CT shall only be used to exclude patients who are unlikely to 
benefit from treatment (See Exclusion criteria in Section 5). If other, advanced imaging 
examinations are used (see sections 6.3 and 6.4, below), findings on such examinations shall not 
influence the decision to include the patient, unless the results of such imaging show that the 
patient should or should not receive thrombolytic treatment, in the opinion of the investigator. 
 
6.3 CT perfusion at selected centres before randomisation (substudy) 
Some centres routinely use CT perfusion in patients with wake-up stroke, and these centres will 
be invited to participate in a substudy of CT perfusion. All CT scans taken in the trial will be sent 
to the Trial Co-ordinating Centre for central, blinded analysis. 
 
6.4 Routine use of MRI before randomisation  
Some centres routinely use MRI perfusion or MRI DWI/FLAIR in patients with wake-up stroke. 
These examinations are not part of the trial, but MRI examinations done of patients included into 
the trial shall be sent to the Trial Coordinating Centre for central, blinded analysis. Extra brain 
scanning (CT perfusion or MRI) must not delay treatment, and we will discourage extra scanning 
if randomisation is delayed more than 20 minutes.  
 
7. Patient inclusion 
7.1 Patient information and consent/assent 
Written, informed consent will be sought from all eligible patients. If the patient can consent but 
cannot sign, non-written consent from the patient (witnessed by non-participating health care 
personnel) can be accepted. If the patient is unable to consent, his/her legal representative and/or 
next of kin can consent on the patient’s behalf depending on national legislation. The regulations 
governing these procedures may vary from jurisdiction to jurisdiction. The procedures for 
obtaining consent will therefore have to be approved for each jurisdiction, and will be described 
in separate standard operating procedures. It is noted that legal restrictions in some EU Member 
States prevent performing any trial-specific interventions (e.g. treatment or trial-specific 
diagnostic procedures without obtaining prior informed consent). 
 
If a patient was unable to give consent at trial entry, but regains capacity to receive information 
and give consent, he/she will be given information as soon as possible and asked whether he/she 
is willing to continue his/her participation in the trial. The patient and his/her legal representative 
will be informed of the right to withdraw from the trial and object to the use of his/her data. 
 
7.2 Randomisation procedure and recording of baseline characteristics 
Patients will be randomly allocated to tenecteplase or control in a 1:1 ratio. Randomisation will be 
performed by central computer over the Internet. The investigators will record patient details via a 
secure web interface before randomisation takes place. Investigators may also contact the Trial 
Coordinating Centre via the 24-hours help-line. 
 
The randomisation procedure will include a standard minimisation algorithm which will ensure 
that the treatment groups are balanced for key prognostic factors, such as sex, age, NIHSS score, 
stroke type (OCSP subgroup), delay to thrombolytic treatment, and use of antiplatelet drugs 
within the past 24 hours. To avoid predictable alternation of treatment allocation, and thus 
potential loss of allocation concealment, patients will be allocated with a probability of 0.80 to the 

 
 
 
 
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treatment group that would minimise the difference between the groups on the key prognostic 
factors. 
 
8. Trial treatment 
Patients will be randomly allocated to open-label tenecteplase plus best standard care or to best 
standard care alone. 
 
8.1 Open-label tenecteplase plus best standard care 
The total dose of tenecteplase is 0.25 mg per kg of body weight (maximum 25 mg). The dose 
shall be given as an intravenous bolus. Patients will be on bed rest in accordance with local 
routine following administration of tenecteplase.  
 
After infusion, vital signs, neurological signs and blood pressure will be monitored every 15 
minutes for two hours, then every 30 minutes for 6 hours, than 60 minutes until 24 hours from the 
start of treatment. Blood pressure should be maintained at or below 180/105 mmHg during the 
first 24 hours. Placement of intra-arterial catheters, indwelling bladder catheters, and nasogastric 
tubes shall be avoided for 24 hours if the patient can be safely managed without them. 
Venipunctures should be performed and monitored carefully. 
 
8.2 Best standard care alone 
Patients randomised to control shall not be given tenecteplase or any other thrombolytic agent. 
 
8.3 Best standard care and other treatments 
Both arms will receive best standard care, including intra-arterial interventions for proximal 
cerebral artery occlusion.63 If the patient is given tenecteplase, then aspirin or other antiplatelet or 
anticoagulant drugs shall not be given until 24 hours after termination of infusion and after the 
control CT brain scan. If the patient was allocated to control, he/she will receive aspirin 300 mg as 
a loading dose as soon as possible after randomisation. After the first 24 hours the recommended 
daily dose of aspirin is 75 mg once daily in both the tenecteplase group and the control group. 
Best standard care during the first week also includes treatments to maintain normal homeostasis 
(temperature, blood glucose, hydration, nutrition), as well as lipid lowering and blood pressure 
lowering drugs, in accordance with clinical guidelines. Clinical examinations, including additional 
CT scans will be performed as clinically indicated. 
 
8.4    Management of serious adverse events related to study treatment 
The management of serious adverse events that can be related to treatment with tenecteplase will 
be guided by specific, detailed standard operating procedures. All patients with neurological 
deterioration will undergo emergency head CT. In case of a major bleeding (e.g. intracranial 
haemorrhage), antifibrinolytic agents or transfusion of cryoprecipitate, fresh frozen plasma or 
platelets may be indicated and should be considered. The investigator should institute any 
supplementary investigations of serious adverse events based on their clinical judgement of the 
likely causative factors. This may include seeking further opinion from a specialist in the field of 
the adverse event. 
  
 

 
 
 
 
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9. Visits and examinations from trial entry till end of follow-up 
The duration of the follow-up period is 3 months. During this period, all patients should be treated 
according to standard clinical guidelines, at the discretion of the clinician. Table 2 gives an 
overview of all visits and examinations in the trial. 
 
Table 2. Scheme for visits and examinations 
 
Days 
Months 
1 
2 
7 
3 
Clinical visit 
x 
 
x 
 
Routine blood analysis 
x 
 
x 
 
CT + CT angiography (if possible) before randomisation 
x 
 
 
 
CT perfusion, at selected centres 
(x) 
 
 
 
CT + CT angiography (if possible) 24±6 hrs after randomisation 
 
x 
 
 
Centralised follow-up 
 
 
 
x 
Day 1 is the day of entry into the trial. *Day 7 or day of discharge, whichever occurs first. 
 
9.1 Visits and examinations during stay in hospital 
Clinical visits: These will be performed on day 1 (at baseline) and day 7 (or on the day of 
discharge, whichever occurs first). NIHSS will be performed at both times. ECG will be 
performed on day 1. Clinical events will be recorded on day 7 (or on the day of discharge). 
Outcome assessors will be blinded to the assigned treatment group. 
 
Routine blood analysis will be performed on day 1 (at baseline) and day 7 (or on the day of 
discharge, whichever occurs first). Tests on day 1 will include: haemoglobin, creatinine, blood 
glucose, platelets, prothrombin time/INR, aPTT, TT and/or eucarin clotting time if it is suspected 
that the patient is taking direct thrombin inhibitors or direct factor Xa inhibitors. 
 
Non-contrast CT and CT angiography (if possible): All patients will undergo non-contrast CT and 
CT angiography (if possible) before randomisation, and again at 24±6 hours after randomisation. 
CT angiography at 24±6 hours will only be performed in patients with a positive finding on CT 
angiography before randomisation. 
 
CT perfusion will be performed at selected centres. 
 
9.2 Centralised follow-up via telephone or mail at 3 months (90 ±7 days) 
At 90±7 days, the Trial Coordinating Centre will contact patients (or their carers) via telephone 
(alternatively, via postal mail), blinded to the treatment that the patients received. We may also 
need to contact the patients’ general practitioners and local hospitals to get information from the 
patients’ medical records. We will record information about clinical events, functional status 
(mRS), activities of daily living (BI), health-related quality of life (EuroQol), and cognitive status 
(MMSE, TICS-M). 
9.3 Long-term follow-up by record linkage with central registries 
We will collect data up to three years from trial entry, by using record linkage with data from 
central registries such as national patient registries and the cause-of-death registries in the 
participating countries. 
 

 
 
 
 
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10. 
Effect variables 
10.1 Primary effect variable 
Functional outcome (defined by the mRS) at 3 months 
10.2 Secondary effect variables 
Clinical events: 
 
Any intracranial haemorrhage during follow-up 
 
Symptomatic intracranial haemorrhage by SITS-MOST definition66 
 
Symptomatic intracranial haemorrhage by IST-3 definition1 
 
Parenchymal haemorrhage type 267Stroke progression during follow-up 
 
Recurrent ischaemic stroke during follow-up 
 
Major extra cranial bleeding 
 
Death from all cause during follow-up 
Clinical events are defined in the Appendix. 
 
Clinical outcomes: 
 
Favourable functional outcome at 3 months: mRS 0-1 
 
Good functional outcome at 3 months: mRS 0-2 
 
NIHSS score at 24 hours and day 7 
 
Change in NIHSS score from baseline to 24 hours an day 7 
 
Barthel Index score at 3 months 
 
EuroQol score at 3 months 
 
MMSE score at 3 months 
 
Radiological outcomes 
Radiological outcomes will be defined in a separate imaging protocol. 
 
Health-economic variables: 
Costs related to: 
 
Length of hospital stay 
 
Nursing home care after discharge 
 
Re-hospitalisations during first 3 months 
 
11. 
Analysis and statistical considerations 
11.1 Estimation of sample size 
It is uncertain whether the difference in treatment effect between thrombolysed and non-
thrombolysed wake-up stroke patients will be similar to that of stroke patients with known time of 
onset. We originally based our sample size estimation on the results of a Cochrane systematic 
review of the effect of rt-PA within 4.5 hours of stroke onset,64 assessed as a binary endpoint 
(favourable outcome mRS 0-2 versus mRS 3-6). As the primary endpoint in TWIST is mRS 
across the full ordinal scale (shift analysis), sample size estimation based on ordinal logistic 
regression analysis is more appropriate. 
 
Longer time from stroke onset to treatment in wake-up stroke patients compared to patients with 
known symptom onset is anticipated and this is likely to weaken the treatment effect. However, 
recent trials on patients with wake-up strokes found similar and actually larger differences 
between thrombolysed and non-thrombolysed patients with wake-up stroke/unknown stroke onset 
time. In the largest randomized controlled trial on wake-up strokes, WAKE-UP, with favourable 
outcome defined as mRS 0-1, the difference between thrombolysed and non-thrombolysed 
patients was 11.5%.20 The same difference of 11.5% was also found in a recent meta-analysis of 6 
observational studies on patients with unknown stroke onset time, where favourable outcome was 

 
 
 
 
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defined as mRS 0-2.65 The MRI-based inclusion criteria in WAKE-UP compared to the CT-based 
inclusion in TWIST could lead to a smaller treatment effect in TWIST. Furthermore, selection 
bias in observational studies may result in larger treatment effect than in randomised-controlled 
trials. It is therefore possible that the effect size in TWIST will be closer to the observed effect in 
recent studies on wake-up stroke patients than to the effect in previous studies on patients with 
known time of symptom onset and that the mRS distribution in the control group differs from 
early trials of IV thrombolytic treatment.  
 
Based on these observations, we assume a treatment effect of 10% absolute difference in a binary 
endpoint setting (mRS 0-1 versus mRS 2-6) and a distribution between modified Rankin Scale 
categories similar to that of WAKE-UP with 42% with favourable outcome in the non-
thrombolysed group vs 52% in the thrombolysed group, which corresponds to an odds ratio of 
1.50. We assume a mRS distribution in the control group in six levels (categories 5 and 6 merged) 
as 15%, 27%, 23%, 17%, 13%, 5%, (similar to the control group the WAKE-UP trial). With a 
power of 80%, a two-sided significance level of 5%, and an effect size specified as an odds ratio 
of 1.50 from an ordinal logistic regression model, the estimated sample size is 600. Based on this, 
the revised target is to recruit 600 patients, i.e. 300 patients in each arm. 
 
11.2 Statistical analyses 
A detailed Statistical Analysis Plan has been drawn up and will be published before breaking the 
randomisation code. We will analyse the data according to the intention-to-treat principle. 
Functional outcome will be primarily compared between the study groups by means of ordinal 
logistic regression, after adjustment for covariates. In a secondary analysis, functional outcome 
will be dichotomised (mRS score 0-1 vs. 2-6) and analysed by means of logistic regression. 
 
For clinical events we will estimate odds ratios and 95% confidence intervals using logistic 
regression and estimate hazard ratios and 95% confidence intervals using the Cox proportional 
hazards model, as appropriate. Interactions will be tested using likelihood ratio tests. The risks of 
clinical events will be compared using Kaplan-Meyer survival analyses and log-rank tests. All 
analyses will use 5% two-sided level of significance. 
 
We will perform prespecified subgroup analyses of patients with findings suggestive of penumbra 
(on CT perfusion), of patients included based on presence or absence of DWI/FLAIR mismatch 
on MRI, of patients with proximal cerebral artery occlusion, and of patients with different time 
intervals since awakening, controlling for imbalances in baseline characteristics. Subgroup 
analyses will be prespecified in the Statistical Analysis Plan. Patients treated with intra-arterial 
intervention for proximal cerebral artery occlusion will also be analysed separately, and the 
primary effect variable for this analysis will be cerebral arterial patency before intra-arterial 
intervention. 
 
Any missing components of the baseline NIHSS will be imputed using a regression-based 
technique, using age and all other components of the NIHSS. A worst-case approach will be used 
when handling incomplete dates and times for adverse events. For example, events will be 
assumed to be treatment emergent unless it is clear even from the partial date/time that this is not 
the case.   
 
As subgroup analyses are of an exploratory nature, no adjustment for multiple comparisons will 
be made. 
 

 
 
 
 
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12. 
Trial conduct and practices/procedures 
12.1 Compliance with regulations and guidelines 
The trial will conform to the EU Clinical Trials Directive (2001/20/EC) and national applicable 
regulatory requirements. EudraCT number: 2014-000096-80. ISRCTN number: 10601890. 
ClinicalTrials.gov number: NCT03181360. 
 
12.2 Data protection 
Personal identifiers will not be stored together with clinical information about the patient, but will 
be stored on a separate, password-protected computer with access only for persons in the Trial 
Coordinating Centre who are responsible for central follow-up. The code linking personal 
identifiers with clinical data will be destroyed 15 years after the publication of the primary report, 
of the trial. The procedures for protection of personal information will be approved by the 
Research Ethics Committee, and data protection officials at the University Hospital of North 
Norway. 
 
12.3 Ethical conduct 
The trial will be conducted in accordance with the MRC Guidelines for Good Clinical Practice in 
Clinical Trials, the Council of Europe’s Convention on Human rights and Biomedicine (CETS 
No.: 164), the ICH Harmonized Tripartite Guideline for Good Clinical Practice 
(CPMP/ICH/135/95) and the Declaration of Helsinki (Edinburgh, October 2000). 
 
We will seek informed consent from all patients, and approval from the ethics committees, 
according to local or national regulations. All patients will be monitored carefully during 
treatment and follow-up, and procedures for management of adverse drug reactions, unmasking of 
the trial treatment, and reporting of serious adverse events are given in the protocol. All events 
will be evaluated, and the trial will be monitored, audited, and inspected according to applicable 
regulatory requirements. 
 
12.4 Liability 
Patients are indemnified by insurance specific for patients participating in trials of an 
Investigational Medicinal Product (IMP). Patients are also covered for non-negligent liability by 
the product indemnity provided by the supplier. 
 
12.5 Recording and reporting of serious adverse events 
Serious adverse events will be recorded in the case report forms. In case of unexpected serious 
adverse events, the Trial Coordinating Centre should be notified immediately and within 24 hours 
at the latest, in accordance with the EC guidance document 2011/C 172/01 (“CT-3”). The report 
of the SAE must include an assessment of whether there is a reasonable possibility that the IMP 
caused the event. 
 
Reports of suspected unexpected serious adverse reactions (SUSARs), with all relevant 
information, will be reported in an expedited manner by the Sponsor, to the competent authority, 
the ethics committee, and the Data Monitoring Committee, according to the EU Clinical Trials 
Directive (2001/20/EC) and applicable regulatory requirements. Copies of such reports will be 
sent to the principal investigators and the Sponsor. (Definitions of serious adverse events and 
SUSARs: See Appendix). 
 
Serious adverse events that are expected will not be reported in an expedited manner by the 
Sponsor, for example recurrent ischaemic stroke, intracranial haemorrhage, myocardial infarction, 
or death. These are events that are expected, and that will be reported as end-points in the case 

 
 
 
 
Protocol version/date 200917
16 
report forms. Expected adverse reactions listed in Section 4.8 of the Summary of Product 
Characteristics will also not be reported in an expedited manner.  
 
12.6 Event adjudication 
A central Event Adjudication Committee will evaluate all events, blinded to treatment allocation. 
 
12.7 Monitoring of data quality 
The central computer randomisation system will check eligibility for all patients entered in the 
trial. Data entered over the Internet (at trial entry) and data in case report forms will be checked 
for validity and internal consistency, to ensure high data quality and completeness, and coherence 
with the protocol. Centres with poor standards will be contacted and appropriate measures will be 
taken. During the course of the study the Sponsor will visit all centres at start-up and least once 
for monitoring purposes, with review of the CRF, comparison with source documents, and 
observation of the conduct of the trial and adherence to the Protocol. It will be particularly 
important to check that each patient exists, that a valid consent form is present in the hospital 
notes, to confirm patient information and adherence to the protocol. There will also be frequent 
telephone contacts, with the purpose of facilitating the work and fulfilling the objectives of the 
study. 
 
12.8 Handling of protocol violations and protocol amendments 
The nature and reasons for the protocol violation shall be recorded in the CRF, in the source 
documents and in the monitoring visit report. All of this serious non-compliance will be followed 
up and reported to RA and IEC as per local regulations. In parallel, corrective and/or preventive 
actions will be undertaken and documented, including any retraining of the investigator and site 
staff. All patients who have been included in the trial will be followed up, irrespective of whether 
treatment was discontinued prematurely, or whether the protocol was violated. If treatment 
discontinuations or protocol violations become frequent the Data Monitoring Committee will 
consider whether there is a need to increase the number of patients to be included in the trial.  
 
All important changes to the trial will be specified in protocol amendments. Amendments must be 
approved by the Steering Committee, and the Sponsor has the responsibility to seek approval from 
the competent authorities the ethics committees. Completed and signed protocol amendments will 
be circulated to all those who were on the circulation list for the original protocol. Amendments 
will be listed in chapter 17. 
12.9 Monitoring of effectiveness and safety 
During the course of the trial the Data Monitoring Committee (DMC) will every 6 months 
perform an unblinded review of SAEs, in all patients, and in the prespecified subgroups. The 
DMC will also perform an unblinded interim analysis of the primary efficacy and safety variables 
(functional outcome and intracranial haemorrhage) when half of the patients have been included. 
If, in their view, there is credible evidence of harm, or overwhelming evidence of efficacy, the 
committee will advise the chairman of the Steering Committee. Unless this happens, the Steering 
Committee will remain ignorant of the interim results. 
 
The DMC will also be responsible for monitoring the overall conduct of the trial, and may 
formulate recommendations relating to the selection, recruitment, or retention of participants, or 
their management, or to improving their adherence to protocol-specified regimens, and the 
procedures for data management and quality control. 
 
The DMC will convene before the start of the trial to agree on the exact terms of reference for the 
committee, and will meet annually during the course of the trial. 
. 

 
 
 
 
Protocol version/date 200917
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12.10 Audit and inspection 
All source data and all trial data and material will be made directly available for audit and 
inspection. Source data is all information in original records and certified copies of original 
records that is necessary for the reconstruction and evaluation of the trial. 
 
12.11 Handling of patient data 
All patients will be assigned a unique code number. The patient data will be linked to this number, 
and the patients’ names or other personal identifiers will not be included in the database. The 
patient database will be kept on a separate, secure computer. The code will be stored on another, 
secure computer, and will be deleted 15 years after the results of the trial have been published. 
The trial’s procedures for data protection will conform to the Norwegian applicable regulatory 
requirements, and to the conditions set by the Norwegian Data Inspectorate. 
 
12.12 Handling of the list of treatment codes 
The lists of random treatment codes will be produced by the Statistical Centre at the University of 
Tromsø. Copies of the list will be kept by the person setting up the central randomisation system, 
and the Trial Statistical Centre. The list will be kept secret for all other people involved in the trial 
until the closure of the patient database. 
 
12.13 Screening logs 
Participating centres will be asked to make records of all patients considered for inclusion in the 
trial (screening logs). 
 
12.14 Financial conduct 
Contracts will be agreed between the Sponsor and each of the investigators/institutions. The 
Coordinating Investigator or other people centrally involved in the trial will not have any financial 
or other conflicts of interest in connection to the trial. Patients participating in the trial will be 
reimbursed for their travel expenses. 
 
12.15 Publication and data sharing policy 
The trial will be published in accordance with the CONSORT guidelines and will be presented by 
a writing committee on behalf of the investigators. All participating centres and collaborators will 
be acknowledged in the main publication. The primary results and results of any substudies will 
be presented at international meetings and in public media. 
 
12.16 User involvement 
The Patient Advisory Board consists of representatives from two stroke patient organisations in 
Norway, with Arne Hagen from Norsk forening for slagrammede, and Anne Heimdal from 
Hjerneslag. The board will be consulted in all phases of the study.  
 
13. 
Central trial organisation 
13.1 Sponsors and funding bodies 
The University Hospital of North Norway is the Sponsor of the trial. Address: UNN Tromsø, NO-
9038 Tromsø, Norway. Telephone number: +47 77626000. The Director of Research will act as 
the Sponsor’s legal representative. 
 

 
 
 
 
Protocol version/date 200917
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The trial receives basic funding from the Norwegian Ministry of Health and Care Services 
(Clinical Therapy Research in the Specialist Health Services Research Programme 
(KLINBEFORSK )) and the National Association for Public Health. The trial also receives 
funding from the British Heart Foundation and from the Swiss Heart Foundation. Boehringer-
Ingelheim GmbH will reimburse the Sponsor for the costs of tenecteplase. 
 
13.2 Trial Coordinating Centre  
The Trial Coordinating Centre is based at the Brain and Circulation Research Group at the 
University Hospital of North Norway and the University of Tromsø.  
The Trial Coordinating Centre consists of the following persons: Trial Coordinating Investigator 
and Head of Brain and Circulation Research Group Ellisiv B. Mathiesen, Trial Manager Melinda 
B. Roaldsen, Trial Officer and postdoc researcher Agnethe Eltoft, Assistant Trial Manager Mary-
Helen Søyland, Trial IT Manager David Perry, Trial Research Nurse Tone Bratteng. 
 
Eivind Berge had a central role in the initiation, planning and implementation of the trial and 
was Trial Co-coordinating Investigator until his death in February 2020. 
 
13.3 Trial Statistical Centre 
Tom Wilsgaard (chair), others. 
 
13.4 Executive Committee 
Melinda Roaldsen, Agnethe Eltoft, Ellisiv B. Mathiesen. 
 
13.5 Imaging Scientific Committee 
Agnethe Eltoft, Arnstein Tveiten, representatives from other centres/countries. 
 
13.6 Trial Steering Committee 
Bent Indredavik (Chair), Ellisiv B. Mathiesen, Tom G. Robinson, David Werring, Arnstein 
Tveiten, Jesper Petersson, Erik Lundström, Hanne Krarup Christensen, Jukka Putaala, Janika 
Kõrv, Dalius Jatuzis, Gian Marco De Marchis, Stefan Engelter, Tom Wilsgaard. 
 
13.7 Data Monitoring Committee 
Terje R. Pedersen (Chair), Peter Sandercock, Hans Wedel (statistician). 
 
13.8 Event Adjudication Committee 
To be appointed. 
 
14. 
Time table and end of trial 
First patient included: July 2017 
Last patient included: September 2021 
End of follow-up: December 2021 
Presentation of main results: 2022 
Follow-up by record linkage with central registries: December 2025 
 
The end of the trial is defined as the last visit of the last patient included. 

 
 
 
 
Protocol version/date 200917
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Molina C, Ribo M, Rubiera M, Santamarina E, Delgado-Mederos R, Maisterra O. TNK 
induces faster MCA recanalization and leads to better short- and long-term clinical 
recanalization and leads to better short- and long-term clinical study [Abstract]. Stroke. 
2008;39:563. 
44. 
Smadja D, Chausson N, Joux J, Saint-Vil M, Signate A, Edimonana M, et al. A new 
therapeutic strategy for acute ischemic stroke: sequential combined intravenous tPA-
tenecteplase for proximal middle cerebral artery occlusion based on first results in 13 
consecutive patients. Stroke. 2011;42:1644-1647. 
45. 
Haley EC Jr, Thompson JL, Grotta JC, Lyden PD, Hemmen TG, Brown DL, et al. Phase 
IIB/III trial of tenecteplase in acute ischemic stroke: results of a prematurely terminated 
randomized clinical trial. Stroke. 2010;41:707-711. 
46. 
Parsons MW, Miteff F, Bateman GA, Spratt N, Loiselle A, Attia J, et al. Acute ischemic 
stroke: imaging-guided tenecteplase treatment in an extended time window. Neurology. 
2009;72:915-921. 
47. 
Parsons M, Spratt N, Bivard A, Campbell B, Chung K, Miteff F, et al. A randomized trial 
of tenecteplase versus alteplase for acute ischemic stroke. N Engl J Med. 2012;366:1099-
1107. 
48. 
Huang X, Cheripelli BK, Lloyd SM, Kalladka D, Moreton FC, Siddiqui A, et al. 
Alteplase versus tenecteplase for thrombolysis after ischaemic stroke (ATTEST): a phase 
2, randomised, open-label, blinded endpoint study. Lancet Neurol. 2015;14:368-376. 
49. 
Coutts SB, Dubuc V, Mandzia J, Kenney C, Demchuk AM, Smith EE, et al. 
Tenecteplase-tissue-type plasminogen activator evaluation for minor ischemic stroke with 
proven occlusion. Stroke. 2015;46:769-774. 
50. 
Huang X, Fulton R, Parsons M, et al. Tenecteplase versus Alteplase in acute ischaemic 
stroke thrombolysis: a meta-analysis of individual patient data from randomised studies. 
Abstract - oral presentation. 1st ESO conference, Glasgow, United Kingdom, April 17-19, 
2015.  
51. 
Logallo N, Novotny V, Assmus J, Kvistad CE, Alteheld L, Ronning OM, et al. 
Tenecteplase versus alteplase for management of acute ischaemic stroke (NOR-TEST): a 
phase 3, randomised, open-label, blinded endpoint trial. Lancet Neurol. 2017;16:781-788. 
52. 
Campbell BCV, Mitchell PJ, Churilov L, Yassi N, Kleinig TJ, Dowling RJ, et al. 
Tenecteplase versus alteplase before thrombectomy for ischemic Stroke. N Engl J Med. 
2018;378:1573-1582. 
53. 
Parsons M. Tenecteplase versus Alteplase for Stroke ThrombolysisEvaluation (TASTE) 
trial. https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=363714.  
54. 
A Randomized Controlled Trial of TNK-tPA Versus Standard of Care for Minor Ischemic 
Stroke With Proven Occlusion (TEMPO-2). ClinicalTrials.gov Identifier: NCT02398656.  
55. 
Alteplase-Tenecteplase Trial Evaluation for Stroke Thrombolysis (ATTEST2). 
ClinicalTrials.gov Identifier: NCT02814409. 

 
 
 
 
Protocol version/date 200917
22 
56. 
Kidwell CS, Jahan R, Gornbein J, Alger JR, Nenov V, Ajani Z, et al. A trial of imaging 
selection and endovascular treatment for ischemic stroke. N Engl J Med. 2013;368:914-
923. 
57. 
Thomalla G, Cheng B, Ebinger M, et al. DWI-FLAIR mismatch for the identification of 
patients with acute ischaemic stroke within 4·5 h of symptom onset (PRE-FLAIR): a 
multicentre observational study. Lancet Neurol. 2011;10:978-986. 
58. 
Parsons MW, Pepper EM, Bateman GA, Wang Y, Levi CR. Identification of the 
penumbra and infarct core on hyperacute noncontrast and perfusion CT. Neurology. 
2007;68:730-736. 
59. 
Hopyan J, Ciarallo A, Dowlatshahi D, Howard P, John V, Yeung R, et al. Certainty of 
stroke diagnosis: incremental benefit with CT perfusion over noncontrast CT and CT 
angiography. Radiology. 2010;255:142-153. 
60. 
Ezzeddine MA, Lev MH, McDonald CT, Rordorf G, Oliveira-Filho J, Aksoy FG, et al. 
CT angiography with whole brain perfused blood volume imaging: added clinical value in 
the assessment of acute stroke. Stroke. 2002;33:959-966. 
61. 
Schramm P, Schellinger PD, Klotz E, Kallenberg K, Fiebach JB, Kulkens S, et al. 
Comparison of perfusion computed tomography and computed tomography angiography 
source images with perfusion-weighted imaging and diffusion-weighted imaging in 
patients with acute stroke of less than 6 hours' duration. Stroke. 2004;35:1652-1658. 
62. 
Leiva-Salinas C, Wintermark M, Kidwell CS. Neuroimaging of cerebral ischemia and 
infarction. Neurotherapeutics. 2011;8:19-27. 
63. 
Powers WJ, Rabinstein AA, Ackerson T, Adeoye OM, Bambakidis NC, Becker K, Biller 
J, Brown M, Demaerschalk BM, Hoh B, Jauch EC, Kidwell CS, Leslie-Mazwi TM, 
Ovbiagele B, Scott PA, Sheth KN, Southerland AM, Summers DV, Tirschwell 
DL.Guidelines for the Early Management of Patients With Acute Ischemic Stroke: 2019 
Update to the 2018 Guidelines for the Early Management of Acute Ischemic Stroke: A 
Guideline for Healthcare Professionals From the American Heart Association/American 
Stroke Association. Stroke. 2019 Dec;50(12):e344-e418. 
64. 
Wardlaw JM, Murray V, Berge E, del Zoppo G, Sandercock P, Lindley RL, et al. 
Recombinant tissue plasminogen activator for acute ischaemic stroke: an updated 
systematic review and meta-analysis. Lancet. 2012;379:2364-2372. 
65. 
Zhu RL Xu J, Xie CJ, Hu Y, Wang K. Efficacy and safety of thrombolytic therapy for 
stroke with unknown time of onset: a meta-analysis of observational studies. J Stroke 
Cerebrovasc Dis. 2020;29:104742 
66. 
Wahlgren N, Ahmed N, Davalos A, Ford GA, Grond M, Hacke W, et al. Thrombolysis 
with alteplase for acute ischaemic stroke in the Safe Implementation of Thrombolysis in 
Stroke-Monitoring Study (SITS-MOST): an observational study. Lancet. 
2007;369(9558):275-282 
67.        von Kummer R, Broderick JP, Campbell BCV, Demchuk A, Goyal M, Hill MD, et al. 
The Heidelberg Bleeding Classification: Classification of bleeding events after ischemic 
stroke and reperfusion therapy. Stroke. 2015; 46:2981–2986. 
 
 

 
 
 
 
Protocol version/date 200917
23 
16. 
Appendix 
16.1 Completed and on-going studies of thrombolytic treatment for wake-up 
stroke 
 
 
16.2 Definitions of clinical events 
16.2.1 Serious adverse events 
A serious adverse event is any untoward medical occurrence that, at any dose: 
 
results in death 
 
is life threatening 
 
requires hospitalisation or prolongation of an existing hospitalization 
 
results in disability/incapacity 
 
is a congenital abnormality / birth defect. 
Medical and scientific judgement should be exercised in deciding whether reporting is appropriate 
in other situations, such as important medical events that may not be immediately life-threatening 
or result in death or hospitalisation, but may jeopardise the subject or may require medical or 
surgical intervention to prevent one of the other outcomes listed in the above definition. These 
should also be considered serious. Examples of such events are invasive or malignant cancers, 
intensive treatment in an emergency room or at home for allergic bronchospasm, blood dyscrasias 
or convulsions that do not result in hospitalisation, or development of drug dependency or abuse. 
16.2.2 Death 
Death will be classified according to cause: 
1. Initial stroke 
2. Recurrent stroke 
3. Myocardial infarction 
4. Infection 
5. Other vascular cause (e.g. systemic emboli) 
Trial (clinicaltrials.gov ID) 
Planned 
enrolment 
Participants 
Interventions 
Primary 
outcome 
Recruitment 
period 
WAKE-UP (NCT01525290) 
800 (503 
enrolled) 
Ischaemic 
stroke; 
<4.5 hours after 
awakening; 
MRI 
DWI/FLAIR 
mismatch 
1. IV alteplase 
2. Placebo 
mRS score 
at 90 days 
Nov 2012 – 
June 2017 
WASSABI (NCT01455935) 
90 
Ischaemic 
stroke on wake-
up; <24 h of 
onset; NIHSS 8-
22; 18-80 years; 
Penumbra on 
CT perfusion 
 
1. Best medical care 
2. IV alteplase 
3. IA treatment 
 
mRS score 
at  90 days 
Nov 2011 –  
EXTEND (NCT01580839)* 
*Not a study of wake-up stroke 
per se, but 146 (65%) of the 
included patients had wake-up 
stroke. 
  
400, revised 
target 310 (225 
enrolled) 
NIHSS 4-26; 
Penumbra 
mismatch on 
MRI or CT 
1. IV alteplase 
2. Placebo 
mRS score 
at 90 days 
June 2010 – 
June 2018 
THAWS (NCT02002325) 
300 (131 
enrolled) 
Initial NIHSS 
≥5 and ≤25; 
DWI/FLAIR 
mismatch on 
MRI 
1. IV alteplase 
2. Control 
mRS at 90 
days 
April 2014 – 
July 2018 

 
 
 
 
Protocol version/date 200917
24 
6. Other non-vascular cause (e.g. malignancy, trauma) 
7. Unknown cause 
16.2.3 Recurrent stroke 
Neurological deterioration (increase of ≥2 on NIHSS, after exclusion of other causes for 
neurological deterioration) occurring after 72 hours will be considered as a recurrent stroke. 
Recurrent stroke will be classified as ischaemic, haemorrhagic or unknown (if not documented on 
imaging). 
16.2.4 Symptomatic intracranial haemorrhage (SICH) 
The IST-31 and SITS-MOST66 definition of symptomatic intracranial haemorrhage are used:  
SICH per IST-3: symptomatic intracranial haemorrhage is defined as clinically significant 
deterioration (neurological deterioration, new headache, new acute hypertension, new nausea or 
vomiting, or sudden decrease in conscious level) or death within the first seven days of treatment 
with evidence of either significant brain parenchymal haemorrhage (local or distant from the 
infarct) or significant haemorrhagic transformation (HT) of an infarct on brain imaging. 
SICH per SITS-MOST: SICH is defined as local or remote parenchymal haematoma type 2 on 
the imaging scan obtained 22-36 hours after treatment, plus neurological deterioration, as 
indicated by an increase in National Institutes of Health Stroke Scale (NIHSS) score of ≥4 points 
compared to baseline value or the lowest value between baseline and 24 (± 6) hours, or 
haemorrhage leading to death.  
Parenchymal haematoma type 2 indicates a haematoma exceeding 30% of the infarct, with 
obvious mass effect.67 
16.2.5 Asymptomatic intracranial haemorrhage 
Intracranial haemorrhage on brain MRI or CT without: neurological deterioration, new headache, 
new acute hypertension, new nausea or vomiting or sudden decrease in conscious level.  
 
16.2.6  Neurological deterioration due to index stroke 
The definition is an increase of ≥ 2 points in one or more of the NIHSS (irrespective of 
improvement in any of the other NIHSS sub-scores). When diagnosing neurological deterioration 
due to index stroke, systemic reasons for deterioration, such as drug-induced hypotension, drug-
induced drowsiness, and intercurrent disease should be excluded. No significant haemorrhage 
should be found on post randomization CT or MR scan. 
 
16.2.7  Acute myocardial infarction 
Either one of the following criteria satisfies the diagnosis of myocardial infarction: 
1. Typical rise and gradual fall (troponin) or more rapid rise and fall (CK-MB) of 
biochemical markers of myocardial necrosis with at least one of the following: 
a. ischaemic coronary symptoms; 
b. development of pathologic Q waves on the ECG; 
c. ECG changes indicative of ischemia (ST segment elevation or depression); or 
d. coronary artery intervention (e. g. coronary angioplasty). 
2. Autopsy pathologic findings of an acute myocardial infarction. 
16.2.8  Major extracranial bleeding 
Definition of major extracranial bleeding: Clinically overt bleeding associated with one or more 
of: 
 Transfusion of >2 red cell units of blood 
 A decrease in haemoglobin of 20 g/l (=2 g/dl, = 1.24 mmol/l) 
 Bleeding into retroperitoneum, intraocular space or major joint 
 Bleeding leading to permanent treatment cessation 
 
Otherwise, the bleeding will be characterized as minor. 
 
 

 
 
 
 
Protocol version/date 200917
25 
16.3 Abbreviations 
aPTT  
Activated partial thromboplastin time 
ATTEST 
Alteplase versus tenecteplase for thrombolysis after ischaemic stroke 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
 
Case report form 
CT 
 
Computed tomography 
DWI 
 
Diffusion-weighted imaging  
ECG 
 
Electrocardiography 
ECT 
 
Ecarin clotting time 
EXTEND 
Extending the Time for Thrombolysis in Emergency Neurological Deficits Trial  
FLAIR  
Fluid-attenuated inversion recovery 
IEC 
 
Independent ethics committee 
IMP 
 
Investigational medicinal product 
INR 
 
International normalized ratio  
MMSE  
Mini-mental state examination  
MRI 
 
Magnetic resonance imaging  
MR-RESCUE Mechanical Retrieval and Recanalization of Stroke Clots Using Embolectomy  
mRS 
 
Modified Rankin Scale 
NIHSS  
National Institutes of Health Stroke Scale 
NOR-TEST 
The Norwegian tenecteplase stroke trial 
SSS 
 
Scandinavian Stroke Scale 
TASTE  
Tenecteplase versus Alteplase for Stroke Thrombolysis Evaluation 
THAWS 
THrombolysis for Acute Wake-up and Unclear-onset Strokes With Alteplase at  
0.6 mg/kg Trial 
TT 
 
Thrombin time 
WAKE-UP 
Efficacy and Safety of MRI-based thrombolysis in Wake Up Stroke 
WASSABI 
Wake Up Symptomatic Stroke in Acute Brain Ischemia 
 
17. 
Summary of changes 
17.1 Protocol version/date 160323 
 
Addition of signature page 
 
Addition of section 3.2: Potential benefits and harms of tenecteplase 
 
Change to section 4.2 (Study questions and objectives)  
 
Change to section 5.2 (Exclusion criteria): 
o New exclusion criterion: “NIHSS score >25 or NIHSS consciousness score >2, or  
seizures during stroke onset” 
o Change to wording of exclusion criterion “High risk of bleeding” 
o New exclusion criterion: “Contraindications to tenecteplase” 
o Change to wording of exclusion criterion “Childbearing potential, pregnancy, positive 
pregnancy test, breastfeeding” 
 
Change to section 6.1 (Patient screening) 
 
Change to section 7.1 (Patient information and consent/assent) 
 
Change to section 7.2 (Randomisation procedure and recording of baseline characteristics) 
 
Change to section 8 (Trial treatment) 
 
Change to section 9 (Visits and examinations from trial entry till end of follow-up) 
 
Change to section 11.2 (Statistical analyses) 
 
Change to section 12.2 (Data protection) 
 
Change to section 12.5 (Recording and reporting of serious adverse events) 
 
Change to section 12.7 (Monitoring of data quality) 
 
Change to section 12.8 (Handling of protocol violations) 
 
Change to section 12.9 (Monitoring of effectiveness and safety) 
 
Change to section 12.10 (Audit and inspection) 
 
Change to section 16.2 (Definitions of clinical events) 

 
 
 
 
Protocol version/date 200917
26 
 
Change to section 17 (Summary of changes) 
17.2 Protocol version/date 160421 
 
Change to section 7.1 (Patient information and consent/assent) 
17.3 Protocol version/date 170412 
 
Change to front page, signature page, sections 13.1 (Sponsor) and 13.2 (The Trial 
Coordinating Centre): Change of sponsor 
 
Change to section 12.2 (Data protection) and 12.11 (Handling of data): Change from 
“Norwegian Data Inspectorate” to “Data Protection Officials” 
 
Change to section 12.16 (User involvement): Change in text 
 
Change to section 13.7 (Data Monitoring Committee): New names 
17.4 Protocol version/date 180410 
 
Change to section 5.2 (Exclusion criteria): Reversal of effect of anticoagulant drugs 
 
Change to section 10.2 (Secondary effect variables): New timing 
 
Change to 11.2 (Statistical analyses): Specification of secondary analysis 
 
Change to section 13.1 (Sponsor and Funders), 13.2 (Trial Coordinating Centre), 13.6 (Trial 
Steering Committee): Update 
 
Change to section 14 (Time table): Update 
17.5 Protocol version/date 180704 
 
Section 3: Update on results of recently published relevant studies 
 
Section 5.1: Change to inclusion criterion: “NIHSS score ≥3” 
 
Sections 5.2, 6.1 and 8.1.3: Removal of exclusion criterion: “Patient will be treated with intra-
arterial interventions for proximal cerebral artery occlusion” 
 
Section 10.2: Change to secondary effect variables 
 
Section 11.2: Pre-specification of secondary analyses 
 
Section 13.6: Change to Trial Steering Committee 
17.6 Protocol version/date 200131 
 
Section 14: Update of time table 
17.7 Protocol version/date 200917 
 
Section 5.2: Change to exclusion criteria: the words “or adnexanet” was deleted from the 
exclusion criterion: “Any known defect in coagulation (…)” 
 
Section 11.1: Revision of sample size estimation with calculation based on ordinal shift 
analysis (in line with the primary endpoint in TWIST) and on results from recent studies on 
patients with wake-up stroke 
 
Section 12.16: Change to User involvement (update of members of Patient Advisory Board)  
 
Section 13.1: Correction of Sponsor’s address 
 
Section 13.2: Change to Trial Coordinating Centre 
 
Section 13.4: Change to Executive Committee 
 
Section 13.5: Change to Imaging Scientific Board 
 
Section 13.6: Change to Trial Steering Committee 
 
Section 13.8: Update of Event Adjudication Committee 
 
Section 14: Change to time table 
 
Section 15: Update of references 
 
Section 16.1: Update of table 
 
Section 16.2: Specifications and additions to definitions of clinical events