P3. Abstract Title: A Point-of-Care Assay for Measuring Thrombin Activity in Finger-Prick Whole Blood

Abstract

At sites of vessel injury, thrombin acts as the central mediator of coagulation through catalyzing fibrin clot formation and platelet activation. Thus, numerous commercially available small molecule chromogenic and fluorogenic substrates have been developed to evaluate thrombin activity. These substrates are limited in that they detect both active thrombin and thrombin bound to its inhibitor α2-macroglobulin, have limited specificity for thrombin and can only be used in plasma samples because of signal quenching. But plasma is not reflective of physiological settings because of the lack of red blood cells which can promote coagulation. Furthermore, assays of plasma require anticoagulation using chelating agents followed by recalcification using supraphysiological concentrations of calcium to initiate coagulation. In addition to calcium ions, chelating agents used as anticoagulants also sequester magnesium and zinc ions which alter coagulation. Thus, divalent cations present in physiological settings are removed in plasma assays. Thus, we designed a novel thrombin substrate that allows for thrombin activity assessment in whole blood. The thrombin substrate consists of a mAmetrine and tTomato fluorescence resonance energy transfer (FRET) pair linked by a thrombin recognition sequence (LVPRGVNL). In addition, the thrombin recognition sequence is flanked by short, antiparallel beta sheets that are linked to the FRET pair through a zipper made of arginine and glutamic acid residues. This design allows it to accommodate long thrombin recognition sequences that enhance specificity and ignore inactive thrombin bound to α2-macroglobulin. Thrombin activity is assessed by evaluating for the loss of FRET. Expressed in E.coli, the thrombin substrate contains two hexahistidine tags on its N-terminus and a calmodulin binding protein tag on its C-terminus. Purification based on these tags ensures that only intact substrate is purified; the tags are then removed by a specific bacterial protease. The purity and integrity of the substrate was confirmed using SDS-PAGE and FRET efficiency assessment. The purified substrate demonstrated specificity for thrombin over factor Xa and activated protein C in buffer containing calcium. To determine whether this thrombin substrate can be used as a point-of-care test of thrombin generation, blood (20 µL) obtained from separate finger pricks from the same healthy donor was incubated with the thrombin substrate and reagents (vehicle, APTT reagent, dabigatran) and evaluated in a portable fluorometer. The fluorometer (StellarCase-Fluoro analyzer) uses a 405 nm LED as excitation, a diffraction grating, a PC tablet and is powered by a lithium battery and enclosed in a rugged case. The change in fluorescence emission was analyzed with Excel to obtain a thrombin generation curve. Compared to blood alone, APTT reagent significantly shortened the time to peak thrombin generation. When dabigatran was added to the blood simultaneously with APTT reagent, the time to peak thrombin generation was increased. These results are consistent with published literature using plasma assays that require larger amounts of blood and centrifugation, suggesting that our substrate can be used as a point-of-care thrombin generation test using a single drop of blood. In conclusion, we have developed a thrombin sensor that has specificity for thrombin compared to related proteases and can measure thrombin activity in whole blood. This sensor is currently being developed into a point-of-care test of anticoagulants and has potential to help our understanding of hemostasis through evaluating thrombin generation in whole blood in varied scenarios.