Z-Gly-Gly-Arg-AMC acetate

Detection of thrombin activity based on Z-Gly-Gly-Arg-AMC acetate^[1]

This protocol only provides a guideline, and should be modified according to your specific needs.

1.Solution preparation

(1) Dissolve the Z-Gly-Gly-Arg-AMC acetate solid in an appropriate amount of DMSO to prepare a 50mM stock solution. Store stock solution at -20°C and avoid repeated freezing and thawing.

(2) Dilute the appropriate Z-Gly-Gly-Arg-AMC acetate stock solution in PBS to prepare a 2.5mM working solution. The working solution should be prepared immediately and used up promptly.

(3) Take an appropriate CaCl₂ solution and dissolve in the detection buffer (containing 20mM HEPES, 145mM NaCl, pH 7.5) to prepare the activation buffer.

2. Thrombin generation assay

(1) Mix the plasma with the Z-Gly-Gly-Arg-AMC acetate stock solution at a volume ratio of 4:1, and place the mixture in the wells of a flat-bottom 96-well plate. Add 100µl of the mixture to each well. Add 20µl of activation buffer to each well to trigger the clotting reaction. The final concentration of Z-Gly-Gly-Arg-AMC acetate is 400µM.

(2) Fluorescence is continuously measured at 37°C using a fluorescence plate reader (Ex/Em=355/460nm) for a period of 30min with a read every 3min.

3. Notes for Attention

(1) Z-Gly-Gly-Arg-AMC acetate is photosensitive and the operation process should be light-protected.

(2) To avoid bubbles, the reading should be taken within 10 seconds after the activation buffer (containing CaCl₂) is added.

References:
[1] Tarandovskiy I D, Artemenko E O, Panteleev M A, et al. Antiplatelet agents can promote two-peaked thrombin generation in platelet rich plasma: mechanism and possible applications[J]. PLoS One, 2013, 8(2): e55688.