The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over.

The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K(m) and k(cat), of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa-Arg substrates. CPM had the highest catalytic efficiency (k(cat)/K(m)) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k(cat) and K(m) of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over.

Comparative substrate specificity study of carboxypeptidase U (TAFIa) and carboxypeptidase N: development of highly selective CPU substrates as useful tools for assay development.

BACKGROUND: Measurement of procarboxypeptidase U (TAFI) in plasma by activity-based assays is complicated by the presence of plasma carboxypeptidase N (CPN). Accurate blank measurements, correcting for this interfering CPN activity, should therefore be performed. A selective CPU substrate will make proCPU determination much less time-consuming. METHODS: We searched for selective and sensitive CPU substrates by kinetic screening of different Bz-Xaa-Arg (Xaa=a naturally occurring amino acid) substrates using a novel kinetic assay. RESULTS: The presence of an aromatic amino acid (Phe, Tyr, Trp) resulted in a fairly high selectivity for CPU which was most pronounced with Bz-Trp-Arg showing a 56-fold higher k(cat)/K(m) value for CPU compared to CPN. Next we performed chemical modifications on the structure of those aromatic amino acids. This approach resulted in a fully selective CPU substrate with a 2.5-fold increase in k(cat) value compared to the commonly used Hip-Arg (Bz-Gly-Arg). DISCUSSION: We demonstrated significant differences in substrate specificity between CPU and CPN that were previously not fully appreciated. The selective CPU substrate presented in this paper will allow straightforward determination of proCPU in plasma in the future.

A role for procarboxypepidase U (TAFI) in thrombosis.

The maintenance of the equilibrium between coagulation and fibrinolysis is crucial for normal haemostasis. In contrast, pathologic consequences of imbalance manifest tendencies of bleeding or thrombosis. Procarboxypeptidase U (proCPU, TAFI) is recognized as an important link between the coagulation system and fibrinolysis. Following activation by thrombin (IIa), carboxypeptidase U (CPU) exerts an antifibrinolytic effect by abolishing the cofactor function of partially degraded fibrin in plasminogen (Pg) activation. This review article focuses on the role of the proCPU/CPU system in the balance between fibrin deposition and removal. How a disturbed system can lead to a higher thrombotic tendency is discussed, while CPU inhibition as a new drug target for fibrinolytic therapy is extensively reviewed.

A rapid and sensitive assay for the quantitation of carboxypeptidase N, an important regulator of inflammation.

BACKGROUND: Carboxypeptidase N is a plasma zinc metallocarboxypeptidase which is constitutively expressed in the liver and was identified as the enzyme responsible for inactivating bradykinin and kallidin by removing the C-terminal arginine. Because CPN can cleave the C-terminal arginine of C3a, C4a and C5a it is often referred to as anaphylatoxin inactivator. Markedly reduced levels of circulating CPN are associated with recurrent angioedema and abnormal cutaneous polymorphonuclear cell infiltration. METHODS: In this paper we describe a fast kinetic coupled enzymatic assay for the sensitive measurement of carboxypeptidase N activities in serum samples. The assay makes use of the excellent CPN substrate Benzoyl-L-Alanyl-L-Arginine. RESULTS: This novel assay is very fast, easy to perform and combines good reliability and reproducibility with excellent correlation with the HPLC-assisted assay (r=0.927; n=140). CONCLUSION: The presented assay can be used for high throughput screening of this important regulator of inflammation in clinical plasma or serum samples.

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.

Carboxypeptidase U (TAFIa) decreases the efficacy of thrombolytic therapy in ischemic stroke patients.

INTRODUCTION: Thrombolytic therapy improves clinical outcome in patients with acute ischemic stroke but is compromised by symptomatic intracranial hemorrhage and an unpredictable therapeutic response. In vitro and in vivo data suggest that activation of procarboxypeptidase U (proCPU) inhibits fibrinolysis. AIMS: To investigate whether the extent of proCPU activation is related to efficacy and safety of thrombolytic therapy in ischemic stroke patients. METHODS: In twelve patients with ischemic stroke who were treated with intravenous (n=7) or intra-arterial (n=5) thrombolysis, venous blood samples were taken at different time points before, during and after thrombolytic therapy. ProCPU and carboxypeptidase U (CPU, TAFIa) plasma concentrations were determined by HPLC. The maximal CPU activity (CPU(max)) and the percentage of proCPU consumption during thrombolytic therapy were calculated. The efficacy and safety of the thrombolytic therapy were assessed by evolution of the clinical deficit, recanalisation, final infarct volume, thrombolysis-induced intracranial hemorrhage and mortality. RESULTS: No correlations between CPU(max) or proCPU consumption and patient or stroke characteristics were found. However, CPU(max) is associated with evolution of the clinical deficit and achieved recanalisation. ProCPU consumption is related to the risk of intracranial hemorrhage, mortality and final infarct volume. CONCLUSIONS: Irrespective of patient and stroke characteristics, CPU(max) and proCPU consumption during thrombolytic treatment for ischemic stroke are parameters for therapeutic efficacy and safety. Further evaluation of the clinical applicability of these parameters and further investigation of the potential role for CPU inhibitors as adjunctive therapeutics during thrombolytic treatment may be of value.

Measurement of procarboxypeptidase U (TAFI) in human plasma: a laboratory challenge.

BACKGROUND: The importance of carboxypeptidase U (CPU) as a novel regulator of the fibrinolytic rate has attracted much interest during recent years. CPU circulates in plasma as a zymogen, proCPU, that can be activated by thrombin, thrombin-thrombomodulin (T-Tm), or plasmin. Given that the proCPU concentration in plasma is far below its K(m) for activation by the T-Tm complex, the formation of CPU will be directly proportional to the proCPU concentration. A low or high proCPU plasma concentration might therefore tip the balance between profibrinolytic and antifibrinolytic pathways and thereby cause a predisposition to bleeding or thrombosis. CONTENT: To measure plasma proCPU concentrations, different methods have been developed based on 2 different principles: antigen determination and measurement of CPU activity after quantitative conversion of the proenzyme to its active form by addition of T-Tm. The major drawbacks that should be kept in mind when analyzing clinical samples by both principles are reviewed. CONCLUSIONS: proCPU is a potential prothrombotic risk factor. Evaluation of its relationship with thrombosis requires accurate assays. Many assays used in different clinical settings are inadequately validated, forcing reconsideration of conclusions made in these reports.