Hidden Potential of Highly Efficient and Widely Accessible Thrombolytic Staphylokinase
Abstract
Stroke burden is substantially increasing but current therapeutic drugs are still far from ideal. Here we highlight the vast potential of staphylokinase as an efficient, fibrin-selective, inexpensive, and evolvable thrombolytic agent. The emphasis is escalated by new recent findings. Staphylokinase nonimmunogenic variant was proven noninferior to alteplase in a clinical trial, with decreased risk of intracranial hemorrhage and the advantage of single bolus administration. Furthermore, our detailed kinetic analysis revealed a new staphylokinase limiting bottleneck whose elimination might provide up to 1000-fold higher activity than the clinically approved alteplase. This knowledge of limitations unlocks new possibilities for improvements that are now achievable by the community of protein engineers who have the required expertise and are ready to transform staphylokinase into a powerful molecule. Collectively, the noninferiority and safety of nonimmunogenic staphylokinase together with the newly identified effectivity limitation make staphylokinase a perfect candidate for further exploration, modification, and advancement to make it the next-generation widely accessible thrombolytic drug effectively treating stroke all around the world, including middle- and low-income countries.
With 6.5 million deaths every year, stroke remains the second-leading cause of death and the third-leading cause of death and disability worldwide.1,2 The only thrombolytic therapy currently approved globally utilizes a recombinant tissue-type plasminogen activator, known as alteplase. Despite higher fibrin selectivity than its predecessors streptokinase and urokinase, alteplase exhibits a lower than 50% recanalization rate, neurological side effects, and an increased risk of intracranial hemorrhage.3,4 Alteplase-induced hemorrhage rates were reported to be ≈3% to 6% higher compared with control groups and the risk of fatal hemorrhage within 7 days increased by >2%.5,6
There have been numerous efforts to improve the properties of alteplase by various modification strategies.7 However, it has been 35 years since alteplase initial approval and none of the clinical trials has ever led to a breakthrough in stroke treatment with markedly improved thrombolysis efficiency, including clinical trials with a genetically modified alteplase, named tenecteplase.8 Tenecteplase exhibits increased half-life, improved fibrin selectivity, and higher inhibition resistance compared with alteplase, and it can be administered as a single bolus. However, tenecteplase still suffers from imperfect properties such as insufficient thrombolytic efficiency, low recanalization rate, and retained risk of intracranial hemorrhage.9
An alternative highly fibrin-specific thrombolytic agent staphylokinase was first described in 1948 and produced in 1983.7,10 Its bacterial origin, structure simplicity, and small size make its production easier and significantly less expensive than that of alteplase (Figure). As pointed out in Stroke, >80% of stroke cases arise in low- and middle-income countries. Mortality rates and disability-adjusted life-years lost are >3-fold higher in low-income countries, positioning cost-effectiveness among crucial drug characteristics.14,15 This is further supported by the fact that the price of alteplase has doubled over the last decade.16 Despite promising results of both animal testing17 and an initial randomized coronary patency trial,18 which showed comparable efficiencies of alteplase and staphylokinase, further trials were discontinued owing to the discovery that staphylokinase was highly immunogenic.
The reluctance to use staphylokinase might change with the latest clinical trial by Gusev and coworkers, who tested a nonimmunogenic staphylokinase variant.13 This variant was previously developed by Collen and coworkers and was proven to induce >200-fold lower titers of antibodies in patients while preserving its thrombolytic efficiency and high fibrin specificity (Figure).12 The recent results of the clinical trial concluded that the nonimmunogenic staphylokinase variant was noninferior to alteplase for patients with acute ischemic stroke. Intracranial hemorrhage developed in only 3% of nonimmunogenic staphylokinase-treated patients compared with 8% of patients treated with alteplase (P=0.087). Within 90 days, 4% more people died in the alteplase group (14% versus 10%; P=0.32).13 The outcome of the trial further showed several advantages of staphylokinase over alteplase, including cost-effectiveness and bolus administration, making it suitable for use in ever increasing attention mobile stroke units recently emphasized in Stroke.19 Although some concerns about the design of this clinical trial have been raised,20 the main outcome and prospective thrombolytic effectiveness and safety of nonimmunogenic staphylokinase remained relevant.
The enormous potential of staphylokinase has been further spotlighted by our recent advanced kinetic analysis.11 We showed that the net value of its thrombolytic activity is 10 000-fold higher than originally determined by conventional analysis with approximations. This is due to the identification of new limiting preceding steps, overall making staphylokinase display lower thrombolytic efficiency. Such a finding is a game-changer in the field of thrombolytic drug development: removing the uncovered main bottleneck by well-established methods of protein engineering21 is plausible and might provide a staphylokinase variant that is up to 1000-fold more effective than alteplase (Figure). Moreover, its distinct mode of action might allow for its effective application in a combination thrombolytic therapy that was proven to exhibit a synergistic effect.22 These crucial mechanistic observations pave the way for the development of next-generation thrombolytic drugs and change the paradigm by positioning staphylokinase at the forefront of other thrombolytic proteins.
In summary, favorable efficiency and safety outcomes of the clinical trial of nonimmunogenic staphylokinase13 combined with unprecedented biological activity uncovered by advanced enzyme kinetics11 make this thrombolytic protein an excellent target for improvement endeavors. The small staphylokinase protein devoid of complicated structural features is perfectly suited for further enhancement using molecular modifications. The community of protein engineers has the necessary know-how, computational tools, and laboratory technologies21 to tailor staphylokinase properties and make it a highly efficient, safe, and widely accessible thrombolytic drug.
Acknowledgments
M. Toul contributed to writing the original draft, review, editing, visualization, and project administration. J. Mican, V. Slonkova, D. Nikitin, M. Marek, and D. Bednar contributed to review and editing. J. Damborsky and Z. Prokop contributed to writing the original draft, review, editing, and resources. All authors approved the final version of the article to be published.
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Published online: 30 August 2022
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This work was supported by the Czech Ministry of Education (INBIO – CZ.02.1.01/0.0/0.0/16_026/0008451; ENOCH – CZ.02.1.01/0.0/0.0/16_019/0000868; EXCELES Neuro – LX22NPO5107) and Grant Agency of Czech Republic (20-15915Y).
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- Successful thrombolysis in an elderly patient with a large-scale hemispheric brain infarction, Clinical Medicine (Russian Journal), 102, 8, (661-664), (2025).https://doi.org/10.30629/0023-2149-2024-102-8-661-664
- Split intein-mediated backbone cyclization enhances the stability and activity of staphylokinase, a potent fibrin-selective plasminogen activator, International Journal of Biological Macromolecules, 275, (133448), (2024).https://doi.org/10.1016/j.ijbiomac.2024.133448
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- Ursolic Acid Ameliorated Neuronal Damage by Restoring Microglia-Activated MMP/TIMP Imbalance in vitro, Drug Design, Development and Therapy, Volume 17, (2481-2493), (2023).https://doi.org/10.2147/DDDT.S411408
- C-terminal lysine residues enhance plasminogen activation by inducing conformational flexibility and stabilization of activator complex of staphylokinase with plasmin, Archives of Biochemistry and Biophysics, 743, (109671), (2023).https://doi.org/10.1016/j.abb.2023.109671
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