Tissue Plasminogen Activator in Stroke Treatment PHM142 Fall 2018 Instructor: Ms. Maya Latif Coordinator: Dr. J. Henderson Tissue Plasminogen Activator in Stroke Treatment By: Judy Ung, Laurie Lam, Portia Li, Samantha Robertson PHM 142 October 2, 2018

Tissue plasminogen activator (tPA) Involved in the breakdown of blood clots Serine protease responsible for catalyzing conversion of plasminogen to plasmin Synthesized by vascular endothelial cells and secreted into plasma We will begin with a brief introduction to Tissue Plasminogen Activator (tPA). tPA is an enzyme involved in the breakdown of blood clots to prevent them from becoming too large and occluding the blood vessel. It is a Serine protease that is responsible for catalyzing the conversion of Plasminogen, a zymogen, into Plasmin, it’s active form And it is synthesized by vascular endothelial cells and secreted into plasma (Hé́bert et al., 2015)

Role of tPA in Blood Coagulation Accumulation of fibrin → clot Plasmin is responsible for fibrinolysis tPA: plasminogen (inactive) → plasmin (active) Moving onto the role of tPA in blood coagulation, or more specifically in the process of fibrinolysis. During blood coagulation, blood clots are formed by an accumulation of fibrin. The surface of fibrin offers binding sites for optimal contact between components of the fibrinolytic system, notably plasminogen and tPA. tPA converts inactive plasminogen to active plasmin, and the active form then goes on to dissolve the clot. The stimulatory effect of fibrin surfaces ensures high concentration of both plasminogen and tPA, and thus Plasmin, at fibrin deposits and localizes plasmin activity. (Gravanis & Tsirka, 2008) (Diapharma, 2018)

Structure of tPA 5 domains: 2 kringle domains (K1, K2) - binding of tPA to plasminogen Finger domain (F) - binding of tPA to fibrin Epidermal growth factor-like domain (EGF) - quick hepatic clearance Serine protease domain (P) - active site for cleavage Contains Ser, His, Asp residues Cleaves Arg561-Val56 bond in plasminogen Human t-PA is a 68 kDa serine protease composed of 5 distinct domain structures with autonomous functions There are 2 kringle domains (K1, K2). The K2 domain is reported to be involved in the capacity of tPA to bind plasminogen The finger domain (F) is involved in the binding of tPA to fibrin The epidermal growth factor-like domain is responsible for activating the epidermal growth factor receptor; and is implicated in rapid hepatic clearance of molecule The active site for cleavage of plasminogen to plasmin is found in the serine protease domain, composed of the amino acids His, Asp and Ser. Histidine and Serine are polar amino acids and Aspartate is negatively charged. Though the 3 residues are situated far apart in the primary structure of the protein, once folded, they are in close proximity and thus form the active site. This region cleaves the Arg561-Val562 bond in plasminogen (since arginine is a positively charged amino acid) and allows for the conversion to occur. (Hé́bert et al., 2015)

tPA in Stroke Treatment Annually, 15 million people suffer from stroke worldwide tPA is prominent therapeutic used to treat ischemic stroke What Is an Ischemic Stroke? When a clot occludes a blood vessel supplying the brain Lack of blood flow and oxygen to brain tissue Irreversible tissue damage Annually, 15 million people suffer from stroke worldwide. tPA (tissue plasminogen activator) is a prominent therapeutic amongst a small selection of drug therapies used to treat ischemic stroke. What is an ischemic stroke? Ischemic strokes occur when a blood clot occludes a blood vessel supplying blood to the brain. Since the brain tissue is very metabolically active, this lack of blood flow and oxygen to the brain tissue leads to significant damage -> often leads to tissue necrosis (Wood, 2018) (Gravanis & Tsirka, 2008)

Why tPA? tPA is a fibrinolytic drug that induces successful recanalization of occluded blood vessels while making sure the surrounding region is viable MOA: breaks down blood clots via conversion of plasminogen to plasmin Activity is regulated by fibrin-binding, which increases its catalytic efficiency (unlike uPA, other plasminogen activator) tPA has been proven to unblock blood vessels via fibrinolysis while making sure the surrounding region is still viable Diagram: outlines the mechanism through which tPA activates plasminogen and results in the breakdown of fibrin, which was outlined by Judy earlier. tPA activity is regulated by fibrin-binding, which increases its catalytic efficiency, which does not occur with the other type of plasminogen activator (urokinase) which has a similar function in the human body (Chhabra, 2014) (Gravanis & Tsirka, 2008)

tPA: A Brief History Primary studies: NINDS study (1995): positive effect ECASS study (1995): positive but more moderate effect FDA (1996): approved for treatment of AIS (acute ischemic stroke) in its intravenous recombinant form (rtPA or alteplase) within 3 h of onset of stroke symptoms Pharmacokinetic and pharmacodynamic properties: Short half-life (5-10 min) Higher fibrin specificity compared to other fibrinolytic drugs (SK and UK) Modifications made to recombinant form to enhance PK and PD properties (increase short half-life and increase fibrin specificity) 2 of the most prominent studies include: · NINDS study (1995): one of the first studies to examine tPA as a form of stroke treatment - Had 2 independently powered trials that both showed clinical benefit from tPA usage · ECASS study (1995): showed a beneficial but more moderate effect-> the effect was more evident with the group that got treated within the first three hours after stroke onset · FDA (1996): approved for tPA for treatment of AIS (acute ischemic stroke) in its intravenous recombinant form (rtPA or altepase) within 3 h of onset of stroke symptoms So far, it’s the only FDA- approved treatment for ischemic strokes to this date PK and PD properties: - tPA has a relatively short half-life in the bloodstream, which is about 5-10 minutes - compared to other fibrinolytic drugs tPA has higher fibrin specificity and is therefore able to dissolve clots with less breakdown of circulating fibrinogen that occurs with the other drugs (such as streptokinase and urokinase) (Gravanis & Tsirka, 2008)

Clinical Significance of tPA Gold standard for treating ischemic stroke Reduces degree of injury to brain 30% more likely to have minimal/no disability after 3 months compared to placebo tPA has been used as a first-line treatment for ischemic stroke since it was approved by Health Canada in 1999. Ischemic stroke accounts for 87% of all stroke cases. When administered within a certain window of time, tPA can reduce the severity of and the damage caused by the cerebrovascular event; in turn, this leads to better prospects for survival and recovery. A study published in the New England Journal of Medicine found that patients given tPA were at least 30% more likely to have minimal or no disability at 3 months after the event compared to the placebo group. (Heart and Stroke Foundation of Canada, 2018) (The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995)

Limitations Short timeframe for success (within 4.5 hours of symptom onset) Only 3 to 5% of stroke patients can receive tPA As I hinted on the previous slide, tPA is only effective in treating stroke for a small window of time: no more than 4.5 hours from the onset of stroke symptoms. This limits the number of patients that are eligible for treatment, which is estimated to be only 3 to 5% of all stroke sufferers. For this reason, it is critical to identify a stroke using the FAST acronym and seek treatment immediately for the best possible chance at a full recovery. (Roth, 2011)

Complications Increased risk of intracerebral hemorrhage Occurs in 6% of patients tPA cleaves matrix metalloproteinases (MMPs) → increase in BBB permeability, leakage, bleeding Unfortunately, the use of tPA to treat stroke is associated with a 10-fold increase in the risk of intracerebral hemorrhage which can cause further injury and lead to death. This occurs in about 6% of patients given tPA and carries a mortality rate close to 50%. The reason for this correlation is that the administered tPA can cleave zymogens other than plasminogen. In this case, it activates matrix metalloproteinases (MMPs) which degrade tight junctions in endothelial cells that form the blood-brain barrier. This leads to leakage, edema, and bleeding in the brain. Due to this life-threatening adverse effect, all stroke patients are carefully assessed before being given tPA to ensure that the benefits outweigh the risks. (Ning et al., 2010) (Lakhan, Kirchgessner, Tepper & Leonard, 2013)

Alteplase Brand name: Activase Thrombolytic drug class & fibrinolytic agent Indication: acute ischemic stroke Increased risk of bleeding leads to contraindications Developed by Genentech Inc. in 1987 In Canada: supplied by Hoffmann-La Roche Limited Other uses Alteplase, another name for tPA, is the drug used to treat acute ischemic stroke. The brand name of this drug is Activase. Alteplase is a fibrinolytic agent in the thrombolytic drug class, and it is produced using recombinant DNA technology. Like other drugs in its class, Alteplase increases the risk of bleeding. The indication for use is the management of acute ischemic stroke for adults. The successful outcome would be improvement in neurological recovery and reducing disability following the stroke. The contraindications for Alteplase are related to the increased risk of bleeding, they include but are not limited to, a hemorrhage type stroke, recent intracranial or intraspinal surgery, recent head trauma, previous strokes, uncontrolled hypertension, seizure upon stroke onset and active internal bleeding. The development of Activase was completed in 1987 in San Francisco by the pharmaceutical company Genentech Incorporated. In Canada it is under the Hoffman-La Roche Limited umbrella. Alteplase is also used in other treatments, such as Activase for acute myocardial infarction and cathflo for the restoration of central venous access devices. (Genentech Inc., 2010)

Administration Intravenous administration 3 hour treatment window Reconstituted immediately before use 50 mg and 100 mg vials 1 mg/mL Activase solution Blood pressure is monitored tPA is administered intravenously through the arm. It can be an effective treatment if it is administered within 3 hours of the onset of the stroke. This window can be extended to 4.5 hours in certain cases. Before it is administered the possibly of an intracranial hemorrhage needs to be ruled out using a CT scan. Activase comes as a lyophilized powder, in 50 mg and 100 mg vials. The vials also come with a diluent for reconstitution, which should be done immediately prior to use, by adding sterile water for injection. The result is a transparent 1mg/ml Activase solution that can be colorless to a pale yellow color. Throughout the duration of treatment blood pressure should be monitored and controlled. If bleeding occurs, as it is the most common complication, Activase should be stopped immediately. There is little evidence on re-administration, it is advised to take caution if you do re-administer. (Genentech Inc., 2010)

Dosage 0.9 mg per kg Maximum dose of 90 mg 10% of total dose is administered in the first minute Remaining 90% is administered over one hour The exact dosage for Activase is 0.9 mg/kg of bodyweight. However, this is not to exceed 90 mg, which is the maximum dosage of this drug. 10% of the total calculated dose is administered in one minute and the remainder of the dose is infused over 60 minutes. (Genentech Inc., 2010)

Presentation Summary tPA converts plasminogen to plasmin through catalytic cleavage Plasmin breaks down fibrin clots in the blood Ischemic stroke is caused by clots that occlude blood flow to the brain → can lead to permanent tissue damage or death tPA (alteplase) is the main therapeutic for ischemic stroke treatment in Canada and the US → increases chances of survival with minimal/ no disability Findings from several studies support the positive effect of tPA in stroke patients Drug must be administered intravenously within 3 to 4.5 hours of symptom onset Adverse effect: intracranial hemorrhage tPA cleaves and activates MMPs: degradation of tight junctions essential to BBB → increased permeability → leakage → edema → bleeding in the brain If bleeding occurs during administration, treatment is stopped immediately

References Chhabra, N. (2014). Mechanism of action of tPA [Digital image]. Retrieved from https://usmle.biochemistryformedics.com/immediate-treatment-of-acute-mi/ Diapharma. (2018). Tissue Plasminogen Activator (tPA). Retrieved from https://diapharma.com/tissue-plasminogen-activator- tpa/#The%20major%20components%20of%20the%20fibrinolytic%20system Genentech Inc. (2010). Frequently asked questions about Activase (alteplase) in acute ischemic stroke. Retrieved from https://www.heart.org/idc/groups/heart- public/@wcm/@mwa/documents/downloadable/ucm_438896.pdf Gravanis, I., & Tsirka, S.E. (2008). tPA as a therapeutic target in stroke. Expert Opin Ther Targets, 12(2). http://doi.org/10.1517/14728222.12.2.159 Hé́bert M, et al. (2015). The story of an exceptional serine protease, tissue-type plasminogen activator (tPA). Revue neurologique, 172(3), 186-197. http://dx.doi.org/10.1016/j.neurol.2015.10.002 Lakhan, S. E., Kirchgessner, A., Tepper, D., & Leonard, A. (2013). Matrix metalloproteinases and blood-brain barrier disruption in acute ischemic stroke.Frontiers in Neurology, 4(32), 1-15. https://doi.org/10.3389/fneur.2013.00032 Ning, M., Sarracino, D. A., Buonanno, F. S., Krastins, B., Chou, S., McMullin, D., … Lo, E. H. (2010). Proteomic protease substrate profiling of tPA treatment in acute ischemic stroke patients: a step toward individualizing thrombolytic therapy at the bedside. Translational Stroke Research, 1(4), 268-275. https://doi.org/10.1007/s12975-010-0047-z Roth, J. M. (2011). Recombinant tissue plasminogen activator for the treatment of acute ischemic stroke. Proceedings (Baylor University Medical Center), 24(3), 257-259. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124916/pdf/bumc0024-0257.pdf The Heart and Stroke Foundation of Canada. (2018). Stroke medications. In Heart and Stroke Foundation. Retrieved from http://www.heartandstroke.ca/stroke/treatments/medications The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. (1995). Tissue plasminogen activator for acute ischemic stroke. The New England Journal of Medicine, 333(24), 1581-1587. https://doi.org/10.1056/NEJM199512143332401 Wood, D. (2018). Hemorrhagic vs. Ischemic Stroke [Digital image]. Retrieved from https://www.cancercarewny.com/content.aspx?chunkiid=11530