Plasma procarboxypeptidase U in men with symptomatic coronary artery disease.

Procarboxypeptidase U (proCPU) is the plasma precursor of carboxypeptidase U (CPU, carboxypeptidase R. plasma carboxypeptidase B or activated thrombin-activatable fibrinolysis inhibitor, TAFIa). CPU removes C-terminal lysine residues that act as plasminogen binding sites from partially degraded fibrin, thereby down-regulating plasminogen activation and fibrinolysis. The present study was carried out as a pilot study to examine whether the plasma proCPU concentration is related to the presence of coronary artery disease (CAD) and/or to levels of established risk indicators for CAD, in a case-control study of 110 men requiring coronary artery bypass grafting (CABG) because of stable angina pectoris. The preoperative plasma proCPU level in the CABG patients was significantly higher than in population-based controls (1029 +/- 154 vs. 974 +/- 140 U/L, p <0.05). In addition, in a subset of the patients (n = 31 ) the proCPU concentration, which was significantly lower on the third postoperative day (-17 +/- 10%), had increased significantly on the sixth day (+14 +/- 12%) after surgery, compared with the preoperative level. In both patients and controls, proCPU concentration was strongly and positively associated with factor VII amidolytic activity and protein C activity, suggesting a common mechanism modulating the plasma levels of these proteins. Otherwise, statistically significant correlations with proCPU were group-specific. In the patients, proCPU correlated significantly with plasma fibrinogen and protein S. In the controls, proCPU correlated significantly with concentrations of cholesterol in plasma. VLDL and LDL. In addition, proCPU correlated significantly with C-reactive protein and haptoglobin levels in the controls only, indicating that also inflammatory mechanisms are involved in the regulation of plasma proCPU. These results suggest that a mechanism exists by which fibrinolytic function is impaired in a manner that is likely to result in more stable fibrin deposits and increase the risk of precocious CAD as well as early occlusion of venous bypass grafts.

Activation of plasma procarboxypeptidase U in different mammalian species points to a conserved pathway of inhibition of fibrinolysis.

Carboxypeptidase U (CPU, EC 3.4.17.20) is a recently described basic carboxypeptidase which circulates in plasma as the zymogen procarboxypeptidase U (proCPU). In the current study, we report on the presence of the proCPU/CPU system in different mammalian species--pig, guinea pig, dog, mouse, rabbit, rat and human. The proCPU concentration, determined as carboxypeptidase activity following thrombin-thrombomodulin activation, ranged from 255 U/l (mouse) to 5051 U/l (pig). When the CPU activity is generated during controlled in vitro coagulation by recalcifying citrated plasma, consistently lower activities were found compared to thrombin-thrombomodulin activation. These data indicate that in all species studied the mechanism for activation of proCPU is present. We demonstrate that in all species studied the addition of PTCI--a CPU inhibitor--results in a marked reduction of the lysis time. Albeit the presence of proCPU, the mechanism of activation during coagulation and the substantial reduction of the clot lysis time in the presence of PTCI point to a conserved inhibitory pathway of fibrinolysis.

Immunological assay for the determination of procarboxypeptidase U antigen levels in human plasma.

The 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 microg/ml (SD 2.5 microg/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.

Local proCPU (TAFI) activation during thrombolytic treatment in a dog model of coronary artery thrombosis can be inhibited with a direct, small molecule thrombin inhibitor (melagatran).

To test the hypothesis that the direct thrombin inhibitor, melagatran is able to inhibit local pro-carboxypeptidase U (proCPU) activation that occurs during thrombolytic treatment, t-PA alone, or in combination with melagatran, was given to dogs with a coronary artery thrombosis. Blood samples from the great cardiac vein and aorta were collected at baseline, during thrombus formation, throughout the t-PA+/-melagatran infusion and during the patency period, for analysis of CPU activity using a novel assay. A higher CPU activity in venous compared to arterial blood (V-A difference) indicates CPU activation in coronary vessels. Efficacy was assessed by determination of time to lysis, duration of patency and blood flow during patency. Dogs (n = 26) were randomized to receive either 1) t-PA, 1 mg/kg as an intravenous 20-min infusion; 2) t-PA as in group 1, +melagatran bolus, 0.3 mg/kg, followed by a 3-h infusion (0.15 mg/kg per h); 3) sham-operated but no coronary thrombus, and administered t-PA as for Group 1. All groups had similar baseline characteristics. Significant increases in CPU activity were observed in Groups 1 and 2 during thrombus formation, with V-A differences of 5.5 and 4.5 U/L, respectively. No significant V-A difference was observed in the sham-operated group. CPU activity increased in Group 1 during the t-PA infusion (V-A difference 15.9 U/L), whereas the V-A difference in Group 2 decreased to 2.6 U/L following melagatran treatment. These results demonstrate that melagatran attenuates generation of CPU in the coronary circulation. The mechanism is probably indirect, via inhibition of thrombin-mediated activation of proCPU.

Proteolytic activation of purified human procarboxypeptidase U.

Carboxypeptidase U (CPU, EC 3.4.17.20) is a recently described basic carboxypeptidase which circulates in plasma as an enzymatically inactive precursor procarboxypeptidase U (proCPU), also known as plasma carboxypeptidase B precursor or thrombin activatable fibrinolysis inhibitor (TAFI). The activation of the zymogen proceeds through a proteolytic cleavage at Arg-92. The active form - CPU - is able to retard the initial phase of fibrinolysis by cleaving C-terminal lysine residues exposed on fibrin partially degraded by the action of plasmin. These C-terminal lysine residues are essential for the high affinity binding of plasminogen to fibrin and the subsequent activation to plasmin. In this report, the activation of purified human proCPU was studied using trypsin and some key proteases of the coagulation and fibrinolytic cascade, i.e., kallikrein, plasmin and thrombin. The most efficient activation is obtained in the presence of thrombin in complex with thrombomodulin. After in vitro activation, CPU is unstable at 37 degrees C (T(1/2)=15 min). Its stability can be improved dramatically using lower temperatures.

Carboxypeptidase U at the interface between coagulation and fibrinolysis.

In 1988, Hendricks et al. first reported on the presence of carboxypeptidase U (U refers to the unstable nature of the enzyme) in human serum. One decade later, the importance of carboxypeptidase U (CPU) in the regulation of fibrin clot dissolution is well documented. CPU circulates in plasma as an inactive zymogen, proCPU, that is converted to its active form during coagulation and fibrinolysis. CPU cleaves off C-terminal lysine residues exposed on fibrin partially degraded by the action of plasmin. Because these C-terminal lysine residues are important for upregulating the fibrinolytic rate, CPU thus slows down fibrinolysis.

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, procarboxypeptidase 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 activation of procarboxypeptidase U by thrombin-thrombomodulin, the resulting enzyme activity cleaves p-OH-Hip-Arg and the generated p-OH-hippuric acid is converted by hippuricase to p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by NaIO4 forms the quinoneimine dye. The absorbance of the latter dye is determined at 506 nm in a microtiter plate reader. A mean value of 620 U/l was found, with a CV of 3.0% within-run and 4.3% between-run. The assay showed a good correlation with the activities observed using a HPLC assay as reference method (n = 25, r = 0.979). The presented method enables the routine analysis of large sample pools in clinical setting.

Isolation and sequence analysis of a human cDNA clone (XPNPEPL) homologous to X-prolyl aminopeptidase (aminopeptidase P).

A novel human cDNA (XPNPEPL) encoding a protein of 623 amino acids exhibiting 44% sequence identity and 62% sequence similarity to pig kidney X-prolyl aminopeptidase (aminopeptidase P; EC 3.4.11.9) was obtained by reverse transcription/polymerase chain reaction of phytohemagglutinin-stimulated lymphocyte mRNA. Conserved sequences were found with the prokaryotic X-prolyl aminopeptidase encoding gene (pepP). The human gene translation product exhibits a high sequence homology to the Schizosaccharomyces pombe chromosome I hypothetical protein C22G7.01c and to the S. cerevisiae ORF y11029w. Northern blot analysis indicates an ubiquitous expression of the human XPNPEPL sequence.

Assay of procarboxypeptidase U, a novel determinant of the fibrinolytic cascade, in human plasma.

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.

Subregional mapping of the human lymphocyte prolyl oligopeptidase gene (PREP) to human chromosome 6q22.

Prolyl oligopeptidase is a large monomeric proline specific serine endopeptidase, the activity of which correlates well with different stages of depression. We have subregionally mapped human lymphocytic prolyl oligopeptidase (PREP) by FISH using a cosmid probe. The probe mapped to the long arm of chromosome 6, and the signal clustered in band q22.