Fast homogeneous assay for plasma procarboxypeptidase U

Carboxypeptidase U (EC 3.4.17.20, CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circulates as an inactive zymogen, procarboxypeptida se U, which becomes active during the process of coagulation. We developed a high throughput method on microtiter plates for the determination of the procarboxypeptidase U concentration in human plasma samples. Following acti vation of procarboxypeptidase U by thrombin-thrombomodulin, the resulting e nzyme activity cleaves p-OH-Hip-Arg and the generated p-OH-hippuric acid is converted by hippuricase to p-hydroxybenzoic acid and glycine. Finally, ox idative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by NaIO4 f orms the quinoneimine dye. The absorbance of the latter dye is determined a t 506 nm in a microtiter plate reader. A mean value of 620 U/I was found, w ith a CV of 3.0% within-run and 4.3% between-run. The assay showed a good c orrelation with the activities observed using a HPLC assay as reference met hod (n = 25, r = 0.979). The presented method enables the routine analysis of large sample pools in clinical setting.