Fast homogeneous assay for plasma procarboxypeptidase U.

Area of Science:

• Biochemistry
• Hematology
• Clinical Chemistry

Background:

• Carboxypeptidase U (CPU), also known as TAFIa, is a key regulator of fibrinolysis.
• CPU circulates as an inactive zymogen, procarboxypeptidase U, activated during coagulation.
• Accurate quantification of procarboxypeptidase U is crucial for understanding its role in hemostasis.

Purpose of the Study:

• To develop and validate a high-throughput microtiter plate assay for determining procarboxypeptidase U concentration in human plasma.
• To establish a reliable method for routine clinical analysis of procarboxypeptidase U levels.

Main Methods:

• Procarboxypeptidase U activation using thrombin-thrombomodulin.
• Enzymatic cleavage of p-OH-Hip-Arg and subsequent conversion of p-OH-hippuric acid.
• Colorimetric detection of a quinoneimine dye formed via oxidative coupling.
• Absorbance measurement at 506 nm using a microtiter plate reader.

Main Results:

• The developed assay demonstrated high throughput capability for plasma sample analysis.
• Mean procarboxypeptidase U concentration was determined to be 620 U/l with low intra- and inter-assay coefficients of variation (3.0% and 4.3%, respectively).
• The assay showed excellent correlation (r = 0.979) with a reference HPLC method.

Conclusions:

• The presented microtiter plate assay is a robust and efficient method for quantifying procarboxypeptidase U concentration.
• This assay facilitates routine analysis of large sample cohorts in clinical settings.
• The method provides a valuable tool for research and clinical diagnostics related to fibrinolysis and coagulation.