Specific determination of plasmatic thrombin activity

Area of Science:

• Biochemistry
• Hematology
• Clinical Chemistry

Background:

• Thrombin is a critical enzyme in blood coagulation.
• Measuring thrombin activity is essential for diagnosing hemostasis disorders.
• Existing methods like fibrinogen conversion or chromogenic assays have limitations, including unspecific substrate cleavage in plasma.

Purpose of the Study:

• To optimize a chromogenic substrate assay for accurate thrombin activity measurement in plasma.
• To determine optimal concentrations of chromogenic substrate (HD-CHG-Ala-Arg-pNA) and arginine for minimizing interference.
• To establish a standardized method for quantifying thrombin activity in international units.

Main Methods:

• Optimized final concentrations of chromogenic substrate (HD-CHG-Ala-Arg-pNA) and arginine.
• Utilized purified thrombin and plasma samples (normal and factor II-deficient).
• Activated plasma via tissue factor (TF) or contact phase, measuring absorbance changes at 405 nm.

Main Results:

• Optimal chromogenic substrate concentration is less than 0.6 mM to avoid unspecific cleavage.
• Using <0.4 mM substrate and >800 mM arginine in factor II-depleted plasma prevented significant thrombin generation.
• Circulating thrombin activity in healthy donors (EDTA plasma) was 100 ± 20% of norm (5.5 mIU/mL).

Conclusions:

• The optimized chromogenic assay accurately measures thrombin activity independent of clotting.
• This method allows for reliable standardization of thrombin activity in international units across various biological systems.
• The assay overcomes limitations of traditional methods by minimizing interference from other hemostatic enzymes.