The objective of this work was to develop a high throughput assay for testing in vitro the thrombolytic activity using citrated whole blood samples, and to overcome the limitations of currently available techniques. We successfully developed a method that involves forming halo shaped, tissue factor induced, whole blood clots in 96 well plates, and then precisely measuring the thrombolysis process with a spectrophotometer plate reader. We here describe the implementation of this novel method, which we refer to as halo assay, and its validation with plasmin, urokinase and tissue plasminogen activator at different doses. The resulting data is a highly detailed thrombolysis profile, allowing comparison of different fibrinolytic agents. The time point analysis allows kinetic data to be collected and calculated to determine key parameters such as the activation time and the rate of fibrinolysis. We also assessed the capacity of the model to study the effect of clot maturation time on the fibrinolytic rate, an aspect of thrombosis rather unexplored with currently available methods, but of increasing importance in drug development. This novel thrombolysis assay could be an extremely useful research tool; to study the complex process of thrombolysis, and a valuable translational clinical tool; as a screening device to rapidly identify hypo- or hyper-fibrinolysis.

BackgroundThrombolytic therapy for acute thrombosis is limited by life‐threatening side effects such as major bleeding and neurotoxicity. New treatment options with enhanced fibrinolytic potential are therefore required. Here, we report the development of a new thrombolytic molecule that exploits key features of thrombosis. We designed a recombinant microplasminogen modified to be activated by the prothrombotic serine‐protease thrombin (HtPlg), fused to an activation‐specific anti–glycoprotein IIb/IIIa single‐chain antibody (SCE5), thereby hijacking the coagulation system to initiate thrombolysis.Methods and ResultsThe resulting fusion protein named SCE5‐HtPlg shows in vitro targeting towards the highly abundant activated form of the fibrinogen receptor glycoprotein IIb/IIIa expressed on activated human platelets. Following thrombin formation, SCE5‐HtPlg is activated to contain active microplasmin. We evaluate the effectiveness of our targeted thrombolytic construct in two models of thromboembolic disease. Administration of SCE5‐HtPlg (4 μg/g body weight) resulted in effective thrombolysis 20 minutes after injection in a ferric chloride–induced model of mesenteric thrombosis (48±3% versus 92±5% for saline control, P<0.01) and also reduced emboli formation in a model of pulmonary embolism (P<0.01 versus saline). Furthermore, at these effective therapeutic doses, the SCE5‐HtPlg did not prolong bleeding time compared with saline (P=0.99).ConclusionsOur novel fusion molecule is a potent and effective treatment for thrombosis that enables in vivo thrombolysis without bleeding time prolongation. The activation of this construct by thrombin generated within the clot itself rather than by a plasminogen activator, which needs to be delivered systemically, provides a novel targeted approach to improve thrombolysis.