Enhanced clot lysis by a single point mutation in a reteplase variant

Recombinant tissue-type plasminogen activator (rtPA) is the clot lysis drug approved for clinical use, and is characterised by a short half-life and substantial inactivation by plasminogen activator inhibitor-1 (PAI-1). We previously discovered that a tPA mutation (A419Y) at the protease domain led to enhanced fibrinolysis activity. In the present study, we studied the mechanism of such mutation in enhancing the proteolytic activity, and whether such enhancement persists in reteplase, an United States Food and Drug Administration-approved tPA truncated variant. We constructed and expressed a series of reteplase-based mutants, including rPA^G (glycosylated rPA), rPA^G -Y (with A419Y mutant at rPA^G ), rPA^G -A4 (tetra-alanine mutation at 37-loop of rPA^G ), and rPA^G -A4/Y (with both) and evaluated their plasminogen activation and PAI-1 resistance. Surface plasmon resonance analysis showed that the rPA^G had fibrin affinity comparable to full-length tPA. Moreover, rPA^G -Y had 8·5-fold higher plasminogen activation and stronger tolerance to PAI-1 compared to rPA^G . We also found that the mutations containing tetra-alanine (rPA^G -A4 and rPA^G -A4/Y) had dramatically reduced plasminogen activation and impaired clot lysis. In a pulmonary embolism murine model, rPA^G -Y displayed a more efficient thrombolytic effect than rPA^G . These results identified a novel mutant reteplase variant of tPA with increased fibrinolytic activity, laying the foundation for the development of a new potent fibrinolytic agent.