Acute ischemic stroke is the result of impaired blood supply to the brain, through thrombosis — the pathological accumulation of occlusive clots in blood vessels that embolise downstream to downstream tissues and micro-vasculature. Injections of thrombolysis, in particular with recombinant tissue-plasminogen activator (rt-PA), have been the go-to option since 1996. rt-PA has many merits but it's seriously unsuitable and unsafe in the treatment of patients, and can be better. The knowledge about basic and clinical research and where thrombolytic therapy is today will make it easier to develop new thrombolytics.

Fig.1. Formation of an occlusive arterial thrombosis. (Mackman, et al., 2020)

Ace Therapeutics' strong capabilities and technologies in the field of stroke contribute to the development of novel thrombolytics. We offer comprehensive services covering every stage of the stroke drug development process, from target identification to preclinical evaluation.

Related Services

• Thrombolytics Development for Stroke
• Anticoagulant Drug Development Services for Stroke
• Antiplatelet Drug Development for Stroke

Current Thrombolytics

tPA

tPA is a serine protease that converts plasminogen to active plasmin, and thus fibrin clots. A lot of clinical work has been devoted to alteplase, a recombinant version of tPA that is still the only FDA-approved thrombolytic agent for acute ischemic stroke.

Protein engineering of alteplase

Protein engineering has been employed to enhance the biochemical properties of alteplase, leading to the development of several variants: monteplase (with a mutation Cys84Ser in the EGF domain), lanoteplase (with a mutation Asn117Gln in the K1 domain), pamiteplase (with K1 domain deleted and a mutation Arg275Glu), and amediplase (with K2 domain from tPA and P domain from urokinase). While these variants show a prolonged plasma half-life, they exhibit reduced fibrin specificity.

Urokinase

Urokinase was discovered in human urine in 1947 and is produced by various cells, including vascular endothelial and smooth muscle cells. Urokinase is more affordable than alteplase and is widely used for treating ischemic stroke in developing countries, as well as for deep venous thrombosis and pulmonary embolism in developed countries.

Streptokinase

Streptokinase, discovered in β-hemolytic streptococci in 1933, acts as an indirect plasminogen activator and is approved for treating acute myocardial infarction, pulmonary embolism, and other thrombotic conditions.

Staphylokinase

Staphylokinase is another indirect plasminogen activator produced by Staphylococcus aureus. It generates plasmin primarily on the surface of fibrin clots, minimizing interactions with free plasmin and reducing bleeding complications. Its effectiveness in thrombolysis has been demonstrated in clinical trials for patients with acute ST-elevation myocardial infarction.

Desmoteplase

Desmoteplase is a 50 kDa serine protease found in the saliva of the vampire bat (Desmodus rotundus), discovered in 1974. Desmoteplase exhibits a unique open conformation upon fibrin binding via the F domain, enhancing its plasmin-generating activity by approximately 100,000-fold, which is the highest among known thrombolytics. Modifications, such as substituting the alteplase F-domain with that of desmoteplase and removing the K2 domain, have demonstrated increased fibrin affinity and stimulation effects.

Development of Novel Thrombolytics

Current FDA-approved thrombolytic drugs, such as streptokinase, urokinase, and alteplase, have several limitations, restricting the overall success rates of the treatments. Thus, there is a need to develop safer and more efficacious treatment strategies to improve clot lysis.

Characteristics of "ideal" Thrombolytics

• Rapid Reperfusion: They should provide quick restoration of blood flow in all patients.
• Prevention of Reocclusions: They should minimize the risk of clots reforming.
• High Fibrin Specificity: They should selectively activate clot-bound plasminogen to reduce bleeding complications.
• Long Plasma Half-Life: They should remain in the bloodstream for an extended period to allow for lower dosages and potentially single bolus administration instead of continuous infusion.
• Resistance to Inhibitors: They should be effective against naturally occurring inhibitors in the body.
• Non-Antigenic: They should not provoke immune responses, enabling repeated use without complications.

Methods for Developing Novel Thrombolytics

To develop novel thrombolytics that address the shortcomings of existing agents, several strategies are employed. On the one hand, novel thrombolytics are being developed using more precise design strategies. Research and developmental strategies in this field have moved from deleting and fusing whole domains of proteins to introducing point mutations. Researchers are increasingly using the methods of rational protein design, which are more specific, focused, and reliable. On the other hand, the vastly expanding repertoire of deoxyribonucleic acid sequences and small molecules enables the discovery of novel thrombolytics in biological samples collected from living ecosystems.

Discovery of new molecules

Numerous natural enzymes and small molecules exhibit thrombolytic activity, leading to the development of new thrombolytics. Key sources include bacteria, such as fibrin-specific thrombolytics from Streptococcus uberis, Bacillus, and Stenotrophomonas. Other sources include various eukaryotes like Chlorella algae, venomous snakes, and polychaetes. Thrombolytics from non-mammalian origins often act as direct fibrinolytics, cleaving fibrin instead of activating plasminogen. Advances in next-generation sequencing and gene-mining software are enhancing the discovery of new thrombolytics.

Domain deletion

Alteplase, composed of five exons that code for various functional domains, is associated with both fibrinolysis and neurological side effects, including blood-brain barrier dysfunction and NMDAR-mediated neurotoxicity. The first three domains of alteplase interact with receptors involved in its clearance by endothelial cells and hepatocytes. In contrast, reteplase is a deletion mutant of alteplase that lacks the finger, EGF-like, and kringle 1 domains. This modification results in a longer half-life of 15 minutes and improves its thrombolytic properties by enhancing clot penetrability and reducing fibrin binding, leading to faster and more uniform thrombolysis with fewer occlusions. Additionally, reteplase's lack of glycosylation and fewer disulfide bridges allow for more cost-effective expression using prokaryotic systems.

Protein chimerization

Protein chimerization involves fusing different proteins or their domains to combine multiple functions within a single protein. For example, fibrin specificity can be enhanced by fusing the EGF domains of thrombomodulin or the K2 domain of alteplase with streptokinase. Another innovative approach is the fusion of microplasmin with an activation-specific anti-glycoprotein IIb/IIIa antibody, allowing targeted fibrinolysis by microplasmin, which is less inhibited by α2-antiplasmin in the bloodstream compared to plasmin.

Point mutations

Researchers use point mutations to improve thrombolytic enzymes such as streptokinase, staphylokinase, alteplase and urokinase. For example, streptokinase half-life can be increased by up to 21 times with a certain residue substitution or PEGylation. It takes a few residue modifications and PEGylations to get the staphylokinase from immunegenicity to fibrinolytic performance.

Liposomes and encapsulation

Thrombolytics can be capsulated into liposomes or other nanomaterials for potential ways to improve thrombolytic therapy. Liposomes are lipid bilayers that can hold therapeutic molecules in their envelopes to prolong thrombolytic half-life by insulating it from clearance receptors and inhibiting plasmalemma throughout the body. The half-life of liposomal streptokinase can be extended by 16-fold, while tPA can be prolonged by 21-fold through encapsulation.

Liposomes also provide beneficial pharmacokinetic properties, allowing for targeted therapeutic action at specific times after stroke, thanks to their ability to accumulate via transcellular transport. Additionally, liposomes can mitigate immunogenicity and possess anti-inflammatory effects, especially when loaded with neuroprotective agents like dexamethasone.

Targeting thrombolytics directly to clots can be achieved by decorating liposomes with fibrinogen-mimicking peptides, allowing for high-specificity binding to platelets. This targeted approach enables effective thrombolytic action using lower doses of urokinase, resulting in reduced bleeding times compared to free urokinase.

Moreover, the release of thrombolytics can be controlled using external stimuli like ultrasound or infrared radiation, enhancing their therapeutic effects. Blood cell membrane-based nanoparticles have shown improved efficacy in animal models of ischemic stroke and thrombosis while maintaining coagulation profiles and reducing bleeding complications. Despite the potential benefits, the translation of these encapsulation strategies into clinical practice is still ongoing.

Rational protein design

Deliberate protein design assisted by crystal structure analysis of thrombolytic enzymes has helped to move thrombolytic agents one step further. For example, by learning about the structure of staphylokinase, PEGylation sites that increase its half-life without degrading its activity have been identified. Similarly, the crystal structure of streptokinase bound to microplasmin allows researchers to study and predict the effects of mutations on its activity.

Although the complete structure of alteplase remains unresolved, in silico modeling using low-resolution structures has provided insights into how domain deletions can impact its catalytic activity, as well as the effects of glycosylation and fibrin enhancement. Additionally, the structure of reteplase has been improved through homology modeling based on alteplase, resulting in better solubility and fibrin affinity.

Proteolysis targeting chimeras (PROTACs)

PROTACs represent a novel class of molecules that enable the controlled degradation of specific proteins, offering significant advantages for therapeutic discovery. These bifunctional molecules bind both a protein of interest (POI) and an E3 ligase, forming a ternary complex that leads to the selective ubiquitination and subsequent degradation of the POI via the proteasome. Unlike traditional gene knockout methods, PROTACs do not require genetic manipulation, making them particularly useful for treating acute ischemic stroke.

Fig.2. Scheme depicting the development of novel thrombolytics. (Nikitin, et al., 2021)

Novel Thrombolytics in Clinical Trials

Several novel thrombolytic targets and associated agents are currently being evaluated in clinical trials.

1. Mackman, N., et al. (2020). Therapeutic strategies for thrombosis: new targets and approaches. Nature reviews Drug discovery, 19(5), 333-352.
2. Nikitin, D., et al. (2021). Development and testing of thrombolytics in stroke.Journal of Stroke, 23(1), 12-36.