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The application of clot\u2010dissolving thrombolytic enzymes is a cost\u2010effective therapeutic intervention for these life\u2010threatening 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\u2010life, 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\u00ae\/Actilyse\u00ae) and tenecteplase (Metalyse\u00ae\/TNKase\u00ae). Consistent with previous studies, tenecteplase exhibited increased fibrin selectivity and inhibition resistance, which explains its extended half\u2010life. Our findings reinforce the growing consensus that tenecteplase may be a superior alternative to alteplase for thrombolytic treatment.<\/jats:p>","DOI":"10.1002\/2211-5463.70132","type":"journal-article","created":{"date-parts":[[2025,10,4]],"date-time":"2025-10-04T08:29:50Z","timestamp":1759566590000},"page":"314-330","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Thrombolytic proteins profiling: High\u2010throughput activity, selectivity, and resistance assays"],"prefix":"10.1002","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3707-6899","authenticated-orcid":false,"given":"Martin","family":"Toul","sequence":"first","affiliation":[{"name":"Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science Masaryk University  Brno Czech Republic"},{"name":"International Clinical Research Center St. Anne's University Hospital  Brno Czech 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