A novel biosensor for measuring plasmin activity.

BACKGROUND

Plasmin facilitates fibrinolysis by catalyzing the degradation of fibrin clots. Reliable methods to assess fibrinolysis by measuring plasmin generation are needed in the preclinical and clinical settings.

OBJECTIVES

This study aimed to design, synthesize, and evaluate a fluorescence resonance energy quenching-based plasmin sensor (FPS) protein to monitor plasmin generation.

METHODS

The hydrolysis of FPS was monitored by loss of fluorescence resonance energy quenching to evaluate efficiency and specificity. Plasma-based plasmin generation assays were used to evaluate fibrinolytic cofactors and inhibitors.

RESULTS

Compared with a peptide plasmin sensor, FPS demonstrated higher efficiency for plasmin and was not cleaved by plasmin inhibited by α2-macroglobulin. FPS interacts with plasmin's kringle domain 5. FPS can measure plasmin generation in clotting plasma, which was increased by an inhibitory antibody against α2-antiplasmin and attenuated by aprotinin, tranexamic acid, and fibrin absence. Maximal plasmin generation occurs after peak clot formation. Furthermore, when antiplasmin is inhibited, the presence of plasminogen activator inhibitor-1 can be measured. Unlike peptide sensors, FPS does not inhibit clot lysis. Lastly, plasmin generation measured using FPS is relatively consistent in plasma from healthy volunteers, with only a 2-fold variation in peak plasmin, peak time, and endogenous plasmin potential.

CONCLUSION

FPS may improve the evaluation of fibrinolysis in preclinical and clinical settings, thereby enhancing our understanding of fibrinolytic disorders.