Novel and specific protein-based biosensors for measuring thrombin and plasmin activity

Abstract

At sites of vessel injury, thrombin mediates coagulation by catalyzing fibrin clot formation and platelet activation. Conversely, plasmin facilitates fibrinolysis by catalyzing the degradation of fibrin clots. Thrombin generation is most frequently measured in plasma samples using small molecule substrates, these substrates have low free thrombin efficiency and specificity, cannot measure thrombin’s exosite interactions and have limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells, require anticoagulation and the addition of supraphysiological concentrations of calcium. To overcome these limitations, we have designed and characterized a fluorescence resonance energy quenching (FREQ)-based thrombin sensor (FTS) protein. Compared to small molecule substrates, the FTS demonstrated high specificity for thrombin because it is not cleaved by thrombin inhibited by α2-macroglobulin and interacts with thrombin’s anion binding exosite I. The FTS can effectively measure thrombin generation in plasma and in whole blood. The FTS does not inhibit standard thrombin generation assays. Lastly, FTS-based thrombin generation in non-anticoagulated finger-prick blood is delayed but enhanced compared to citrated plasma. Similarly, plasmin generation is also restricted to plasma samples and measured using the fluorogenic Boc-Glu-Lys-Lys-AMC, which have low free plasmin efficiency and specificity, cannot measure plasmin’s kringle domain interactions and are insensitive to the effects of plasminogen activator inhibitor-1 (PAI-1). Here, we describe the design and characterization of a (FREQ)-based plasmin sensor (FPS) protein that demonstrated high efficiency for plasmin that is not inhibited by α2-macroglobulin when compared to Boc-Glu-Lys-Lys-AMC and interacts with plasmin’s kringle domain 5. The FPS measures plasmin generation in plasma, where it demonstrated greater sensitivity to tranexamic acid compared to Boc-Glu-Lys-Lys-AMC as well as sensitivity to PAI-1 and the effects of fibrin. Therefore, the FTS and FPS will broaden our understanding of thrombin and plasmin generation in ways that are not attainable with current methods.