Practical application of a chromogenic FXIIa assay

Area of Science:

• Biochemistry
• Hematology
• Biomaterials Science

Background:

• Autohydrolysis of blood factor XII (FXII) to FXIIa is a known reaction.
• This autohydrolysis is significantly influenced by the reaction environment, being facile in buffer solutions but negligible in the presence of plasma proteins.

Purpose of the Study:

• To investigate the impact of FXII autohydrolysis on chromogenic assays for FXIIa.
• To develop a reliable chromogenic assay for FXIIa that accounts for autohydrolysis.
• To explore the application of FXII autohydrolysis in sensitive biomaterial hemocompatibility testing.

Main Methods:

• Investigated FXII autohydrolysis kinetics in neat-buffer solutions versus plasma protein presence.
• Compared chromogenic assay results for FXIIa in buffer with traditional plasma-coagulation assays.
• Developed and validated a rate-assay method for FXIIa detection using swamping concentrations of FXII.

Main Results:

• FXII autohydrolysis is a facile reaction in buffer but insignificant in plasma.
• Autohydrolysis causes significant deviations in standard chromogenic FXIIa assays performed in buffer.
• A rate-assay method accommodating autohydrolysis achieves analytical agreement with plasma-coagulation assays.
• Autohydrolysis can amplify FXIIa, enabling sensitive detection of biomaterial-induced FXIIa generation.

Conclusions:

• FXII autohydrolysis must be considered for accurate FXIIa measurement in buffer solutions.
• A modified chromogenic rate-assay method provides reliable FXIIa quantification.
• FXII autohydrolysis offers a promising amplification strategy for highly sensitive biomaterial hemocompatibility assessment.