The decrease in procarboxypeptidase U (TAFI) concentration in acute ischemic stroke correlates with stroke severity, evolution and outcome.

BACKGROUND AND OBJECTIVES: Procarboxypeptidase U (proCPU, TAFI) concentration in plasma is potentially related to thrombotic tendency, and elevated proCPU levels have been reported in ischemic stroke patients. Improved insight into the role of proCPU in acute ischemic stroke is essential for the development of more adequate therapeutics that may include carboxypeptidase inhibitors. In this study we investigated whether the plasma concentration of proCPU and the proCPU kinetic profile in acute ischemic stroke are related to initial stroke severity, stroke evolution in the subacute phase and long-term stroke outcome. METHODS: Plasma concentration of proCPU was assessed in 136 stroke patients at admission (7.5 h after stroke onset), at 24 h, at 72 h and at day 7 after stroke onset. We evaluated the relation between change in proCPU concentrations and (a) stroke severity (patients with TIA vs. stroke patients, NIHSS score at admission), (b) stroke evolution (stroke progression, infarct volume at 72 h), and (c) stroke outcome (mRS score at month 3). RESULTS: ProCPU concentration decreased significantly in the first 72 h after stroke onset and thereafter returned to baseline. This biphasic time course, with its nadir at 72 h, was more pronounced in patients with severe stroke, unfavourable stroke evolution in the first 72 h and poor long-term outcome. CONCLUSIONS: The decrease in proCPU concentration in the first 72 h after stroke onset correlates with more severe stroke, unfavourable stroke evolution, and poor long-term stroke outcome.

Carboxypeptidase U (TAFIa): a new drug target for fibrinolytic therapy?

Procarboxypeptidase U (TAFI) is a recently discovered plasma procarboxypeptidase that upon activation by thrombin or thrombin-thrombomodulin turns into a potent antifibrinolytic enzyme. Its prominent bridging function between coagulation and fibrinolysis raised the interest of many research groups and of the pharmaceutical industry. The development of carboxypeptidase U (CPU) inhibitors as profibrinolytic agents is an attractive concept and possibilities for rational drug design will become more readily available in the near future as a result of the recently published crystal structure. Numerous studies have been performed and many of them show beneficial effects of CPU inhibitors for the improvement of endogenous fibrinolysis in different animal sepsis and thrombosis models. CPU inhibitors combined with tissue-type plasminogen activator (t-PA) seem to increase the efficiency of pharmacological thrombolysis allowing lower dosing of t-PA and subsequently fewer bleeding complications. This review will focus on recently obtained in vivo data and the benefits/risks of targeting CPU for the treatment of thrombotic disorders.

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

OBJECTIVE: To investigate the effects of short-term postmenopausal oral hormone administration on plasma levels of procarboxypeptidase U (proCPU, thrombin-activatable fibrinolysis inhibitor, EC 3.4.17.20), an inhibitor of fibrinolysis, in healthy early postmenopausal women. DESIGN: A prospective, randomized, placebo-controlled study. SETTING: Outpatient clinic of the Department of Obstetrics and Gynaecology. SUBJECTS: Seventy-seven healthy early postmenopausal women were screened of whom 65 were randomized. Analyses were based on 60 participants. INTERVENTIONS: The women received oral micronized oestradiol 2 mg either alone (E2 group, n=16), or sequentially combined with dydrogesterone 10 mg (E2 + D group, n=14) or trimegestone 0.5 mg (E2 + T, n=14), or placebo (n=16) for 12 weeks. MAIN OUTCOME MEASURE: ProCPU concentrations at baseline, and at 4 and 12 weeks of treatment. RESULTS: Four weeks of E2 + T was associated with a significant decrease in the fasting proCPU concentration, which was sustained after 12 weeks [t=0: 636 +/- 57 U L(-1) (mean +/- SD); t=4: 583 +/- 63UL-1; t=12: 589 +/- 48 U L(-1); ANCOVA versus placebo: P=0.011]. The percentage change from baseline versus placebo in this group was -8.4% [95% confidence interval (CI) -15.7 to -1.1] after 4 weeks and -5.9% (95% CI -11.7 to -0.1) after 12 weeks. There were no significant changes versus placebo in the E2 group nor in the E2 + D group. CONCLUSION: Short-term treatment with E2 + T, but not E2 alone or E2 + D, lowers proCPU concentration. These findings add to accumulating evidence suggesting that different progestagens added to oestrogen replacement may differentially affect the risk of arterial and venous disease.

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.

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.

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.

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.

Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen.

A novel basic carboxypeptidase clearly different from carboxypeptidase N has been isolated from human plasma. It circulates as an enzymatically inactive precursor enzyme bound to plasminogen. During fibrinolysis, it can be converted to its active form, carboxypeptidase U, through the action of plasmin. The active enzyme has an apparent molecular weight of 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzes the synthetic peptides hippuryl-L-arginine and hippuryl-L-lysine but, in contrast to other human basic carboxypeptidases, has only a limited esterase activity. After its activation, carboxypeptidase U tends to be very unstable.

Immobilized carboxypeptidase N. A potent bioreactor and specific adsorbent for peptides.

Carboxypeptidase N-Sepharose was prepared by covalent immobilization of purified human plasma carboxypeptidase N. More than 98% of the carboxypeptidase N was immobilized; 42% of the applied activity can be detected on the support. The column has excellent capabilities to quantitatively remove carboxy-terminal basic amino acids from peptides, as is demonstrated using the synthetic peptide substrate hippuryl-L-arginine and the nonapeptide bradykinin, and remains stable for several months. In contrast with apocarboxypeptidase B-Sepharose, apocarboxypeptidase N-Sepharose poorly binds its substrates.

Exopeptidases in human platelets: an indication for proteolytic modulation of biologically active peptides.

The determination in human platelets of four exopeptidases--aminopeptidase P, dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme--by means of fluorometric or liquid chromatography techniques was carried out. The results obtained show that the specific activities of dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme in intact and disrupted platelets are small compared to their specific activities in serum. However, for aminopeptidase P the specific activity of this enzyme is much higher in platelets than in serum. This suggests that circulating platelets may have a significant role as scavengers for circulating peptides containing bonds susceptible for aminopeptidase P.