SummaryCarboxypeptidase U (CPU, TAFIa) recently gained interest as a significant player in dampening the fibrinolytic rate. The aim of this study was to investigate the time course of the generation of CPU activity during coagulation and fibrinolysis using an in vitro clot lysis model in human plasma. A first peak of CPU activity appeared after initiation of the coagulation phase and a second rise in CPU activity was observed during the fibrinolysis. The decrease in the proCPU plasma concentration followed the same trend as the appearance of the CPU activity. The direct thrombin inhibitor inogatran eliminated the CPU generation during coagulation but not during fibrinolysis. Addition of the plasmin inhibitor aprotinin during fibrinolysis resulted in a decrease in CPU activation during the lysis phase. These results demonstrate that proCPU was activated during coagulation by thrombin and during fibrinolysis by plasmin. Addition of a CPU inhibitor before initiation of clotting decreased the clot lysis time as expected. However, addition in the time period between the two peaks of CPU activity had no apparent effect on the clot lysis time.
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.
SummaryThe importance of carboxypeptidase U as a novel regulator of the fibrinolytic rate has attracted a lot of interest recently. In the present work, an ELISA was developed using polyclonal antibodies raised against recombinant proCPU, expressed in DON cells. The assay determines the antigen concentration of the zymogen of carboxypeptidase U, procarboxypeptidase U, in human citrated plasma or EDTA plasma. No interference is observed with plasma carboxypeptidase N. The assay is very reproducible (within-run: 4.6% CV, between-run: 6.8% CV). In a group of 479 healthy individuals the mean proCPU antigen concentration is 13.4 μg/ml (SD 2.5 μg/ml). A good correlation is found with the functional procarboxypeptidase U assay described earlier (r = 0.82, p <0.0001) (Schatteman K, Goossens F, Scharpé S, Neels H, Hendriks D Clin Chem 1999; 45: 807-813). The significant correlation between the proCPU antigen concentration and the 50% clot lysis time stresses its importance as a player in fibrinolysis control.
Abbreviations: CPU, carboxypeptidase U; proCPU, procarboxypeptidase U; TAFI, thrombin activatable fibrinolysis inhibitor; AP, alkaline phosphatase; EDTA, ethylenediamine tetra-acetic acid; Tris, tris-(hydroxyethyl)-aminomethane; PPACK, phenylalanyl-prolyl-chloromethyl ketone; SP, sulphopropyl; HEPES, N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid
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