Thrombolytic proteins profiling: High‐throughput activity, selectivity, and resistance assays

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.