A low bleeding risk thrombolytic agent: citPA5.

Abstract

Alteplase is a cornerstone thrombolytic agent in clinical practice, but presents a potential bleeding risk. Stroke patients need pre-screening to exclude hemorrhagic stroke before using Alteplase. In this study, we develop a new thrombolytic agent citPA5, characterized by an enhanced safety profile and minimal bleeding tendency. A clot lysis agent, named citPA5, is developed based on rtPA with point mutations to completely suppress its proteolytic activity in the absence of fibrin. In the presence of fibrin, citPA5 exhibited significantly higher fibrinolytic activity (a 15.8-fold increase of kcat/Km). Furthermore, citPA5 showed resistance to endogenous fibrinolysis inhibitor, PAI-1, resulting in enhanced potency. In a series of safety evaluation experiments, including thrombelastography (TEG) assay, mice tail bleeding assay, and a murine intracerebral hemorrhage (ICH) model, citPA5 did not cause systemic bleeding or worsen intracerebral hemorrhage compared to Alteplase. This highlights the low risk of bleeding associated with citPA5. Finally, we found that citPA5 effectively improved cerebral blood flow and reduced infarct volume in a carotid embolism-induced stroke (CES) model. This clot lysis agent, citPA5, not only exhibits a low risk of bleeding but also demonstrates highly effective thrombolysis capabilities. As a result, citPA5 shows great potential for administration prior to the classification of stroke types, making it possible for use in ambulances at the onset of stroke when symptoms are identified. The findings presented in this study also suggest that this strategy could be applied to develop a new generation of fibrinolytic drugs that offer greater safety and specificity in targeting fibrin.