Real-Time Fibrinolysis Monitoring of Plasma Annular Clots

Fibrinolysis assessment is critical for diagnosing and managing clinical blood disorders. Currently available viscoelastic testing platforms provide an overview of global coagulation and fibrinolysis profiles but lack fibrinolysis specific assessment of a preformed clot substrate. The lack of a sensitive, standardized testing platform for fibrinolysis assessment can limit risk stratification and the management of blood disorders. We describe herein the plasma annular clot lysis assay. The assay adapts the previously standardized FITC-tagged fibrin annular clot assay to plasma obtained from healthy human donors. Plasmin at concentrations ranging from 200-800 nM was used as a direct thrombolytic to assess fibrinolysis in plasma-derived annular clots. The Maximum Fibrinolysis Rate (V_MFR), FLU200, T90, and MaxFLU were calculated via tracking of the clot digestion curve over time. V_MFR was correlated with plasma thromboelastography (TEG) parameters to compare with the global viscoelastic testing system. Additionally, plasma annular clot digestion was monitored in the presence of pentamidine to assess drug-specific effects on fibrinolysis tracking. Plasma annular clots linearly tracked fibrinolysis with increasing plasmin concentration. V_MFR across multiple tested plasmin concentrations showed a moderate to strong negative correlation (0.58-0.74) with the observed maximum amplitude from plasma TEG. In the presence of pentamidine (75 µM), the assay was sensitive to identifying differences in the V_MFR across treatment groups. Plasma annular clots provide a platform for fibrinolysis evaluation using a patient's own plasma to assess therapeutic dosing clinically in addition to testing novel therapeutics preclinically to further understand mechanistic aspects of fibrinolysis.