Quantitative and Continuous Analysis of ATP Release from Blood Platelets with Firefly Luciferase Luminescence

Summary

An analytical method was devised to determine the entire course of the adenosine-triphosphate (ATP) release from blood platelets based on a continuous measurement of firefly luciferase luminescence. The equation of the release reaction was derived after due consideration of substrate (ATP) consumption and product (oxyluciferin) inhibition on the luciferase reaction as follows: C, = v_t × (K_m/V_max) × (I + I_t/K_i) + I_t where C_t is the total concentration of the released ATP at time t, v_t is the velocity of the luciferase reaction at time t and is directly measured, I_t is the concentration of oxyluciferin at time t, and V_max, K_m and K_i are constants. C, of the gel-filtered platelet suspension (GFP) could be determined by substituting v_t and I_t as functions of t. The effects of albumin and temperature on the reaction were also studied.

Key words

ATP release from blood platelets - Firefly luciferase luminescence - Time-course kinetics - Product inhibition

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