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The Nijmegen Hemostasis Assay: Simultaneous Fluorogenic Measurement of Thrombin and Plasmin Generation in a Single Well
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Specific determination of plasmatic thrombin activity.

T W Stief1

  • 1Department of Clinical Chemistry, University Hospital, Giessen & Marburg, Germany. thstief@med.uni-marburg.de

Clinical and Applied Thrombosis/Hemostasis : Official Journal of the International Academy of Clinical and Applied Thrombosis/Hemostasis
|September 9, 2006
PubMed
Summary

This study optimizes a chromogenic assay for measuring thrombin activity in plasma. The enhanced assay accurately quantizes thrombin levels, independent of clotting, for standardized diagnostics.

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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.