Judith Leurs

Carboxypeptidase U (TAFIa) prevents lysis from proceeding into the propagation phase through a threshold-dependent mechanism

Summary.  In an in vitro clot lysis model in human plasma, carboxypeptidase U (CPU) is generated by thrombin following the coagulation and by plasmin at the later stage of clot lysis. CPU is able to slow down clot lysis by suppressing the cofactor activity of partially degraded fibrin in the plasminogen activation by tissue‐type plasminogen activator (t‐PA). Making use of thrombomodulin and a thrombin inhibitor, the generation of CPU during the in vitro clot lysis can be manipulated both in terms of magnitude and time course. The data obtained demonstrate that CPU affects the clot dissolution through a threshold‐dependent mechanism: as long as the CPU activity remains above the threshold value, lysis is prevented from proceeding into the propagation phase. From the moment the CPU activity drops below this threshold value, the rate of lysis accelerates. This threshold value for CPU activity is dictated by the t‐PA concentration: increasing the t‐PA concentration increases the CPU threshold and vice versa. This implies that the effect of the CPU pathway will become more apparent at a lower fibrinolytic capacity. Our threshold‐based hypothesis indicates that the time course of proCPU activation, the stability of CPU and the t‐PA concentration all play a crucial role in determining the result of the in vitro clot lysis experiment. Furthermore, this hypothesis provides us with new insights into previously published data on the effects of CPU on in vitro clot lysis by high and low t‐PA concentrations.

Development of a Genotype 325–Specific proCPU/TAFI ELISA

Objective—A Thr/Ile polymorphism at position 325 in the coding region of proCPU has been reported. Immunological assays, fully characterized (including genotype dependency), are required for the quantitation of proCPU levels. Methods and Results—We have generated a panel of monoclonal antibodies against human, plasma-derived proCPU. Two combinations exhibiting distinct reactivities were selected for measurement of proCPU in plasma. T12D11/T28G6-HRP yielded values of 10.1±3.1 &mgr;g/mL (mean±SD, n=86; normal donors), and T32F6/T9G12-HRP yielded values of 5.4±3.0 &mgr;g/mL. Grouping according to the 325 genotype demonstrated that T12D11/T28G6-HRP was independent to this polymorphism whereas T32F6/T9G12-HRP revealed a complete lack of reactivity with the Ile/Ile genotype (ie, 0.0±0.0, 4.2±1.7, and 7.3±2.9 &mgr;g/mL for the Ile/Ile, Ile/Thr, and Thr/Thr isoforms, respectively). Commercially available antigen assays appeared to be partially dependent on the 325 genotype (eg, 44±8.9% and 100±30% for the Ile/Ile and Thr/Thr isoforms, respectively). Conclusions—Our data demonstrate that great care should be taken when evaluating proCPU antigen values as a putative causative agent or as a diagnostic risk marker for cardiovascular events.

Carboxypeptidase U (TAFIa): a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease.

Since the discovery of Carboxypeptidase U (CPU) in 1988, considerable information has been gathered about its biochemistry and function in physiological and pathophysiological circumstances. A variety of tools such as assays to measure proCPU and CPU, antibodies raised against (pro)CPU, selective CPU inhibitors and knock-out mice have been developed and are currently being used to explore the role of this metallocarboxypeptidase in different in vivo and in vitro settings. The knowledge that proCPU can be activated by thrombin and plasmin, enzymes with a key function in coagulation and fibrinolysis, and the ability of CPU to remove C-terminal lysine residues has led to the hypothesis that the proCPU/CPU pathway plays a role in the balance between coagulation and fibrinolysis. The maintenance of the equilibrium between coagulation and fibrinolysis is crucial for normal haemostasis and disturbance of this delicate balance can lead either to bleeding tendency or thrombosis. This review provides an update on several aspects of CPU known at the moment, including an extensive overview on the clinical studies performed up till now.

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

Abstract. Post MS, Hendriks DF, van der Mooren MJ, van Baal WM, Leurs JR, Emeis JJ, Kenemans P, Stehouwer CDA (VU university medical center, Amsterdam, The Netherlands; University of Antwerp, Wilrijk, Belgium; and Gaubius Laboratory, TNO‐PG, Leiden, The Netherlands). Oral oestradiol/trimegestone replacement reduces procarboxypeptidase U (TAFI): a randomized, placebo‐controlled, 12‐week study in early postmenopausal women. J Intern Med 2002; 251: 245–251.

Raloxifene reduces procarboxypeptidase U, an antifibrinolytic marker. A 2-year randomized, placebo-controlled study in healthy early postmenopausal women.

Objective The aim of this study was to compare the long-term effects of two dosages of raloxifene with oral hormone therapy (HT; conjugated equine estrogens combined with medroxyprogesterone acetate) on procarboxypeptidase U. Design In a randomized, double-blind, placebo-controlled, 2-year study, 95 healthy, nonhysterectomized, early postmenopausal women received either daily raloxifene 60 mg (n = 24), raloxifene 150 mg (n = 23), HT (conjugated equine estrogens 0.625 mg + medroxyprogesterone acetate 2.5 mg, n = 24), or placebo (n = 24). At baseline and after 6, 12, and 24 months, fasting plasma procarboxypeptidase U concentrations were measured. Results Six months of treatment with raloxifene 60 mg and raloxifene 150 mg were associated with significant decreases in plasma procarboxypeptidase U concentrations, which were sustained after 12 and 24 months. Raloxifene 60 mg: t = 0, 619 ± 89 U/L (mean ± SD); t = 6, 574 ± 87 U/L; t = 12, 571 ± 96 U/L; t = 24, 568 ± 92 U/L; ANCOVA versus placebo, P = 0.026. Raloxifene 150 mg: t = 0, 608 ± 67 U/L; t = 6, 580 ± 73 U/L; t = 12, 578 ± 70 U/L; t = 24, 562 ± 61 U/L; ANCOVA versus placebo, P = 0.039. No significant changes were found in the HT group. Conclusion Long-term treatment with raloxifene reduced procarboxypeptidase U plasma concentrations.

Fast homogeneous assay for plasma procarboxypeptidase U.

Abstract 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.

LETTERS TO THE EDITOR: Fast kinetic assay for the determination of procarboxypeptidase U (TAFI) in human plasma

Semeraro N. Effect of heparin on TAFI-dependent inhibition of fibrinolysis: relative importance of TAFIa generated by clot-bound and fluid phase thrombin. Thromb Haemost 2002; 88: 282–7. 13 Mosnier LO, Buijtenhuijs P, Marx PF, Meijers JC, Bouma BN. Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood 2003; 101: 4844–6. 14 Genser N, Lechleitner P, Maier J, Dienstl F, Artner-Dworzak E, Puschendorf B, Mair J. Rebound increase of plasminogen activator inhibitor type I after cessation of thrombolytic treatment for acute myocardial infarction is independent of type of plasminogen activator used. Clin Chem 1998; 44: 209–14. 15 Meade TW, Ruddock V, Stirling Y, Chakrabarti R, Miller GJ. Fibrinolytic activity, clotting factors, and long-term incidence of ischaemic heart disease in the Northwick Park Heart Study. Lancet 1993; 342: 1076–9. 16 Munkvad S, Gram J, Jespersen J. A depression of active tissue plasminogen activator in plasma characterizes patients with unstable angina pectoris who develop myocardial infarction. Eur Heart J 1990; 11: 525–8. 17 NewbyDE,WrightRA,LabinjohC, LudlamCA, FoxKA, BoonNA, Webb DJ. Endothelial dysfunction, impaired endogenous fibrinolysis, and cigarette smoking: a mechanism for arterial thrombosis and myocardial infarction. Circulation 1999; 99: 1411–5. 18 Newby DE, McLeod AL, Uren NG, Flint L, Ludlam CA, Webb DJ, Fox KA, Boon NA. Impaired coronary tissue plasminogen activator release is associated with coronary atherosclerosis and cigarette smoking: direct link between endothelial dysfunction and atherothrombosis. Circulation 2001; 103: 1936–41.

arboxypeptidase 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.

HMR 3339, a novel selective estrogen receptor modulator, reduces concentrations of procarboxypeptidase U, an inhibitor of fibrinolysis. A randomized, placebo‐controlled study in postmenopausal women

T . E . VOGELVANG,* J . R . LEURS , V. MI JATOVIC ,* J . WILLEMS E , and M. J . VAN D ER MOOREN* Project ‘Aging Women’ and the Institute for Cardiovascular Research-Vrije Universiteit (ICaR-VU), *Department of Obstetrics and Gynecology, VU University Medical Center, Amsterdam, the Netherlands; Department of Medical Biochemistry, University of Antwerp, Wilrijk, Belgium; for the HMR 3339 Research Group

Fast kinetic assay for the determination of procarboxypeptidase U (TAFI) in human plasma: Letters to the Editor

Semeraro N. Effect of heparin on TAFI-dependent inhibition of fibrinolysis: relative importance of TAFIa generated by clot-bound and fluid phase thrombin. Thromb Haemost 2002; 88: 282–7. 13 Mosnier LO, Buijtenhuijs P, Marx PF, Meijers JC, Bouma BN. Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood 2003; 101: 4844–6. 14 Genser N, Lechleitner P, Maier J, Dienstl F, Artner-Dworzak E, Puschendorf B, Mair J. Rebound increase of plasminogen activator inhibitor type I after cessation of thrombolytic treatment for acute myocardial infarction is independent of type of plasminogen activator used. Clin Chem 1998; 44: 209–14. 15 Meade TW, Ruddock V, Stirling Y, Chakrabarti R, Miller GJ. Fibrinolytic activity, clotting factors, and long-term incidence of ischaemic heart disease in the Northwick Park Heart Study. Lancet 1993; 342: 1076–9. 16 Munkvad S, Gram J, Jespersen J. A depression of active tissue plasminogen activator in plasma characterizes patients with unstable angina pectoris who develop myocardial infarction. Eur Heart J 1990; 11: 525–8. 17 NewbyDE,WrightRA,LabinjohC, LudlamCA, FoxKA, BoonNA, Webb DJ. Endothelial dysfunction, impaired endogenous fibrinolysis, and cigarette smoking: a mechanism for arterial thrombosis and myocardial infarction. Circulation 1999; 99: 1411–5. 18 Newby DE, McLeod AL, Uren NG, Flint L, Ludlam CA, Webb DJ, Fox KA, Boon NA. Impaired coronary tissue plasminogen activator release is associated with coronary atherosclerosis and cigarette smoking: direct link between endothelial dysfunction and atherothrombosis. Circulation 2001; 103: 1936–41.