Cardiovascular diseases, including thrombotic events such as ischemic stroke, pulmonary embolism, and myocardial infarction, are among the leading causes of morbidity and disability worldwide. The application of clot-dissolving thrombolytic enzymes is a cost-effective therapeutic intervention for these life-threatening conditions. However, the effectiveness and safety profiles of current drugs are suboptimal, necessitating the discovery of new medicines or the engineering and enhancement of the existing ones. Here, we present a set of optimized biochemical protocols that allow robust screening and the therapeutic potential assessment of thrombolytic biomolecules. The assays provide information on multiple characteristics such as enzymatic activity, fibrinolysis rate, fibrin and fibrinogen stimulation, fibrin selectivity, clot binding affinity, and inhibition resistance. Such detailed characterization enables a rapid and reliable evaluation of candidate effectiveness and provides an indication of biological half-life, associated bleeding complications, and other side effects. We demonstrate the credibility of the methodology by applying it to the two most widely used thrombolytic drugs: alteplase (Activase®/Actilyse®) and tenecteplase (Metalyse®/TNKase®). Consistent with previous studies, tenecteplase exhibited increased fibrin selectivity and inhibition resistance, which explains its extended half-life. Our findings reinforce the growing consensus that tenecteplase may be a superior alternative to alteplase for thrombolytic treatment.

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Brno Czech Republic

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Tsao CW, Aday AW, Almarzooq ZI, Alonso A, Beaton AZ, Bittencourt MS, Boehme AK, Buxton AE, Carson AP, Commodore‐Mensah Y et al. (2022) Heart disease and stroke statistics—2022 update: a report from the american heart association. Circulation 145, e153–e639.

Roth GA, Abate D, Abate KH, Abay SM, Abbafati C, Abbasi N, Abbastabar H, Abd‐Allah F, Abdela J, Abdelalim A et al. (2018) Global, regional, and national age‐sex‐specific mortality for 282 causes of death in 195 countries and territories, 1980–2017: a systematic analysis for the global burden of disease study 2017. Lancet 392, 1736–1788.

Risman RA, Abdelhamid A, Weisel JW, Bannish BE and Tutwiler V (2022) Effects of clot contraction on clot degradation: a mathematical and experimental approach. Biophys J 121, 3271–3285.

Alkarithi G, Duval C, Shi Y, Macrae FL and Ariëns RAS (2021) Thrombus structural composition in cardiovascular disease. Arterioscler Thromb Vasc Biol 41, 2370–2383.

Tawil SE and Muir KW (2017) Thrombolysis and thrombectomy for acute ischaemic stroke. Clin Med 17, 161–165.

Aguiar de Sousa D, von Martial R, Abilleira S, Gattringer T, Kobayashi A, Gallofré M, Fazekas F, Szikora I, Feigin V, Caso V et al. (2019) Access to and delivery of acute ischaemic stroke treatments: a survey of national scientific societies and stroke experts in 44 European countries. Eur Stroke J 4, 13–28.

Nikitin D, Choi S, Mican J, Toul M, Ryu W‐S, Damborsky J, Mikulik R and Kim D‐E (2021) Development and testing of thrombolytics in stroke. J Stroke 23, 12–36.

Baruah DB, Dash RN, Chaudhari MR and Kadam SS (2006) Plasminogen activators: a comparison. Vasc Pharmacol 44, 1–9.

Mican J, Toul M, Bednar D and Damborsky J (2019) Structural biology and protein engineering of thrombolytics. Comput Struct Biotechnol J 17, 917–938.

Rijken DC and Collen D (1981) Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 256, 7035–7041.

Lähteenmäki K, Kuusela P and Korhonen TK (2001) Bacterial plasminogen activators and receptors. FEMS Microbiol Rev 25, 531–552.

Reddy KNN and Markus G (1972) Mechanism of activation of human plasminogen by streptokinase: presence of active center in streptokinase‐plasminogen complex. J Biol Chem 247, 1683–1691.

Toul M, Nikitin D, Marek M, Damborsky J and Prokop Z (2022) Extended mechanism of the plasminogen activator staphylokinase revealed by global kinetic analysis: 1000‐fold higher catalytic activity than that of clinically used alteplase. ACS Catal 12, 3807–3814.

Nikitin D, Mican J, Toul M, Bednar D, Peskova M, Kittova P, Thalerova S, Vitecek J, Damborsky J, Mikulik R et al. (2022) Computer‐aided engineering of staphylokinase toward enhanced affinity and selectivity for plasmin. Comput Struct Biotechnol J 20, 1366–1377.

Toul M, Mican J, Slonkova V, Nikitin D, Marek M, Bednar D, Damborsky J and Prokop Z (2022) Hidden potential of highly efficient and widely accessible thrombolytic staphylokinase. Stroke 53, 3235–3237.

Toul M, Slonkova V, Mican J, Urminsky A, Tomkova M, Sedlak E, Bednar D, Damborsky J, Hernychova L and Prokop Z (2023) Identification, characterization, and engineering of glycosylation in thrombolytics. Biotechnol Adv 66, 108174.

Víteček J, Vítečková Wünschová A, Thalerová S, Gulati S, Kubala L, Capandová M, Hampl A and Mikulík R (2024) Factors influencing the efficacy of recombinant tissue plasminogen activator: implications for ischemic stroke treatment. PLoS One 19, e0302269.

Capstick T and Henry MT (2005) Efficacy of thrombolytic agents in the treatment of pulmonary embolism. Eur Respir J 26, 864–874.

Collen D (1996) Fibrin‐selective thrombolytic therapy for acute myocardial infarction. Circulation 93, 857–865.

Tsikouris JP and Tsikouris AP (2001) A review of available fibrin‐specific thrombolytic agents used in acute myocardial infarction. Pharmacotherapy 21, 207–217.

Zhu Y, Carmeliet P and Fay WP (1999) Plasminogen activator inhibitor‐1 is a major determinant of arterial thrombolysis resistance. Circulation 99, 3050–3055.

Tjärnlund‐Wolf A, Brogren H, Lo EH and Wang X (2012) Plasminogen activator inhibitor‐1 and thrombotic cerebrovascular diseases. Stroke 43, 2833–2839.

Yang Y, Gu B and Xu XY (2023) In silico study of different thrombolytic agents for fibrinolysis in acute ischemic stroke. Pharmaceutics 15, 797.

Marcos‐Contreras OA, Ganguly K, Yamamoto A, Shlansky‐Goldberg R, Cines DB, Muzykantov VR and Murciano J‐C (2013) Clot penetration and retention by plasminogen activators promote fibrinolysis. Biochem Pharmacol 85, 216–222.

Toschi L, Bringmann P, Petri T, Donner P and Schleuning W‐D (1998) Fibrin selectivity of the isolated protease domains of tissue‐type and vampire bat salivary gland plasminogen activators. Eur J Biochem 252, 108–112.

Bringmann P, Gruber D, Liese A, Toschi L, Krätzschmar J, Schleuning W‐D and Donner P (1995) Structural features mediating fibrin selectivity of vampire bat plasminogen activators. J Biol Chem 270, 25596–25603.

Sobel BE (2001) Fibrin specificity of plasminogen activators, rebound generation of thrombin, and their therapeutic implications. Coron Artery Dis 12, 323–332.

Vaughan DE, Goldhaber SZ, Kim J and Loscalzo J (1987) Recombinant tissue plasminogen activator in patients with pulmonary embolism: correlation of fibrinolytic specificity and efficacy. Circulation 75, 1200–1203.

Goldenberg NA, Hathaway WE, Jacobson L and Manco‐Johnson MJ (2005) A new global assay of coagulation and fibrinolysis. Thromb Res 116, 345–356.

Longstaff C and Fibrinolysis TSO (2017) Development of shiny app tools to simplify and standardize the analysis of hemostasis assay data: communication from the SSC of the ISTH. J Thromb Haemost 15, 1044–1046.

Bannish BE, Chernysh IN, Keener JP, Fogelson AL and Weisel JW (2017) Molecular and physical mechanisms of fibrinolysis and thrombolysis from mathematical modeling and experiments. Sci Rep 7, 6914.

Jiao J, Yu M and Ru B (2001) Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding. Biochimica et Biophysica Acta (BBA) 1546, 399–405.

Robbie LA, Bennett B, Croll AM, Brown PA and Booth NA (1996) Proteins of the fibrinolytic system in human thrombi. Thromb Haemost 75, 127–133.

Srinivasan B and Lloyd MD (2024) Dose–response curves and the determination of IC50 and EC50 values. J Med Chem 67, 17931–17934.

Wind T, Jensen MA and Andreasen PA (2001) Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type‐1: implications for antibody‐mediated PAI‐1‐neutralization and vitronectin‐binding. Eur J Biochem 268, 1095–1106.

Reytor Gonzalez ML, del Rivero A and Antigua M (2021) Reviewing the experimental and mathematical factors involved in tight binding inhibitors Ki values determination: the bi‐functional protease inhibitor SmCI as a test model. Biochimie 181, 86–95.

Keyt BA, Paoni NF, Refino CJ, Berleau L, Nguyen H, Chow A, Lai J, Peña L, Pater C and Ogez J (1994) A faster‐acting and more potent form of tissue plasminogen activator. Proc Natl Acad Sci USA 91, 3670–3674.

Thomas GR, Thibodeaux H, Errett CJ, Badillo JM, Keyt BA, Refino CJ, Zivin JA and Bennett WF (1994) A long‐half‐life and fibrin‐specific form of tissue plasminogen activator in rabbit models of embolic stroke and peripheral bleeding. Stroke 25, 2072–2078.

Potla N and Ganti L (2022) Tenecteplase vs. alteplase for acute ischemic stroke: a systematic review. Int J Emerg Med 15, 1.

Paoni NF, Keyt BA, Refino CJ, Chow AM, Nguyen HV, Berleau LT, Badillo J, Peña LC, Brady K and Wurm FM (1993) A slow clearing, fibrin‐specific, PAI‐1 resistant variant of t‐PA (T103N, KHRR 296‐299 AAAA). Thromb Haemost 70, 307–312.