Assay of Procarboxypeptidase U, a Novel Determinant of the Fibrinolytic Cascade, in Human Plasma

Abstract

Background: Procarboxypeptidase U (proCPU) is a novel proenzyme found in human plasma. The active form, carboxypeptidase U (CPU; EC 3.4.17.20), retards the rate of fibrinolysis through its ability to cleave C-terminal lysine residues on fibrin partially degraded by plasmin. This reduces the number of high-affinity plasminogen-binding sites on fibrin. Methods: We developed an assay to determine the proCPU concentration in human plasma. The assay involved quantitative conversion of proCPU to active CPU by thrombin-thrombomodulin, a very efficient activator of proCPU, followed by determination of the enzymatic activity of CPU with the substrate hippuryl-l-arginine, using an HPLC-assisted determination of the released hippuric acid. Using this method, we established a reference interval based on 490 healthy individuals. Results: The mean proCPU concentration, determined after activation of the zymogen in diluted plasma and expressed as CPU activity, was 964 U/L, with a SD of 155 U/L. The population showed a gaussian distribution. However, we noticed important differences related to age and the use of hormone preparations. Conclusions: The sensitivity and precision of the method make it suitable for routine clinical determinations and as a reference procedure.

Comparative study of commercially available procarboxypeptidase U (thrombin‐activatable fibrinolysis inhibitor) assays

“…For the total population (n = 132), a mean proCPU concentration of 993 ± 161 U/L (mean ± standard deviation [SD]) was found when measured with our reference assay. This concentration is in agreement with previously published data for a normal population [6]. The mean proCPU concentration of the study group as measured with the commercial kits corresponded well with the values of a normal population as proposed by the manufacturer(datanotshown).ForActichromeTAFI,however, the proCPU concentrations found were 3.5-fold higher (mean ± SD; 57.5 ± 7.7 lg/mL) than the concentration put forwardintheinsertofthekit(expectedmean:12.0-20.0 lg/mL).…”

Overall haemostatic potential can be used for estimation of thrombin‐activatable fibrinolysis inhibitor‐dependent fibrinolysis in vivo and for possible follow‐up of recombinant factor VIIa treatment in patients with inhibitors to factor VIII

“…Several assays for laboratory determination of TAFI have been developed. TAFI antigen is measured using ELISA [4], while the total amount of pro-TAFI that can be activated is measured using a chromogenic substrate [5]. Assay of clot lysis time (CLT) with and without addition of potato tuber carboxypeptidase inhibitor (PTCI), a specific inhibitor of TAFI, has also been used for an indirect TAFI activity estimation [6].…”

Oral oestradiol/trimegestone replacement reduces procarboxypeptidase U (TAFI): a randomized, placebo‐ controlled, 12‐week study in early postmenopausal women

“…d P < 0.01: paired t-test for within-group changes from baseline. E 2 ¼ micronized 17b-oestradiol 2 mg; D ¼ dydrogesterone 10 mg; T ¼ trimegestone 0.5 mg. 20], but not in women receiving hormonal therapy (oral contraceptives, oestrogen replacement therapy or HRT) [18]. Data concerning proCPU levels in hormone users as compared with nonusers are conflicting: lower proCPU concentrations were found in postmenopausal women using HRT [19], whilst higher levels were described in pre-and postmenopausal women using any kind of hormonal therapy [18].…”

“…It could be speculated that the considerable, unfavourable increase in proCPU in women not using hormonal therapy after the age of 49, compared with those within the age-group 40-49 years [18], is the result of decreasing oestradiol levels in perimenopausal women. Consequently, the lower proCPU levels in HRT users as observed by Juhan-Vague et al [19] could result from the restoration of oestradiol levels.…”