Determination of TAFIa Activity in Plasma by the Hippuryl-Arg-OH Method[1]
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Reagent Preparation: Prepare a 20mM working solution of the Hippuryl-Arg substrate. Prepare working solutions of Thrombin at 5U/mL and Thrombomodulin at 7.5U/mL, then mix them to form a thrombin-thrombomodulin complex. Prepare a 60mM CaCl₂ solution. Prepare an activation buffer (20mM HEPES, 0.15M NaCl, 5mM CaCl₂, pH=7.4). Prepare a 1M HCl solution to stop the reaction. Prepare a 1M NaOH solution. Prepare a 1M phosphate buffer. Prepare a 3% cyanuric chloride solution in 1,4-dioxane.
2. TAFI Activation: Take 15μL of plasma sample and mix it with 12μL of the thrombin-thrombomodulin complex, 8.5μL of 60mM CaCl₂ solution, and 24μL of activation buffer. Incubate the mixture at room temperature for 10 minutes to activate TAFI in the plasma to TAFIa. Subsequently, add 20μL of Hirudin to terminate the activation reaction.
3. TAFIa Activity Detection: To the reaction mixture from the previous step, add 20μL of the 20mM Hippuryl-Arg substrate working solution and incubate at room temperature for 10 minutes. Then, add 20μL of 1M HCl solution to terminate the substrate reaction. Next, sequentially add 20μL of 1M NaOH solution and 25μL of 1M phosphate buffer to the reaction system. Finally, add 60μL of the 3% cyanuric chloride solution in 1,4-dioxane and let it stand for sufficient color development.
4. Detection and Calculation: Centrifuge the color-developed reaction mixture at 14,000g for 2 minutes. Take the supernatant and measure its absorbance at a wavelength of 405nm using a spectrophotometer. All samples and standard curve points should be diluted to 50% with heat-inactivated serum and measured in duplicate.
Precautions:
(1) For your safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Santamaría A, Borrell M, Oliver A, et al. Association of functional thrombin-activatable fibrinolysis inhibitor (TAFI) with conventional cardiovascular risk factors and its correlation with other hemostatic factors in a Spanish population. Am J Hematol. 2004 Aug;76(4):348-52. |
