(62q) Optimization of Fluorogenic Thrombin Generation Assay

Blood coagulation is a cascade of enzymatic reactions leading to the formation of thrombin and eventually clotting plasma. Thrombin is the key coagulation enzyme with many functions: thrombin clots blood plasma by cleaving protein fibrinogen; activates platelets and vascular cells; controls its own production by activating Factors V, VIII, XI and Protein C; and controls fibrinolysis via activation of Factor XIII and TAFI. Traditional assays for blood coagulation function (e.g. APTT and PT) are based on the recording of the end-point clotting time in a blood plasma sample mixed with artificial activators. The clotting tests have limited predictive value and cannot be used for control of many pro- and anti-coagulation treatments. The novel microplate thrombin generation (TG) assay was specifically developed to utilize a physiological activator (low amount of tissue factor) and real-time monitoring of thrombin enzymatic activity via a small fluorogenic peptide substrate for thrombin. The TG assay holds promise as a physiologically relevant and sensitive test of global hemostasis, but as yet is has not been validated. In this study, using Michaelis-Menten kinetics, rate limiting reactions were determined and correlations were made between fluorescent intensely and the amount of active enzyme thrombin generated. We then investigated the artifacts that are introduced by the sub-optimal selection of excitation and emission wavelengths and fluorophore concentrations in a microplate reader experiment. These novel experimental approaches provide useful models for the analysis of blood coagulation kinetics in health and disease.