Evelien Heylen

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

Summary.  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.

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

See also Willemse JL, Brouns R, Heylen E, De Deyn PP, Hendriks DF. Carboxypeptidase U (TAFIa) activity is induced in vivo in ischemic stroke patients receiving thrombolytic therapy. J Thromb Haemost 2008; 6: 200–2.

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.

Carboxypeptidase U (TAFIa) activity is induced in vivo in ischemic stroke patients receiving thrombolytic therapy

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An update on the role of carboxypeptidase U (TAFIa) in fibrinolysis

Since its discovery more than 20 years ago, a lot has been revealed about the biochemistry and physiological behaviour of carboxypeptidase U (CPU). Recent advances in CPU research include the unravelling of the crystal structure of proCPU and revealing the molecular mechanisms for the marked instability of the active enzyme, CPU. The recent development of two highly sensitive assays has cleared the path toward the direct measurement of CPU in circulation or the determination of CPU generation, rather than the measurement of total proCPU concentration in plasma. Finally, since CPU is known to have a prominent bridging function between coagulation and fibrinolysis, the development of CPU inhibitors as profibrinolytic agents is an attractive new concept and has gained a lot of interest from several research groups and from the pharmaceutical industry. These recent advances in CPU research are reviewed in this literature update.

Procarboxypeptidase U (TAFI) contributes to the risk of thrombosis in patients with hereditary thrombophilia

INTRODUCTION It is considered that high plasma levels of procarboxypeptidase U (proCPU or TAFI) can promote the development of thrombosis, but data comparing proCPU levels in thrombophilia carriers and healthy subjects are rather scarce. Moreover, the results of previous studies on the risk of thrombosis related to high proCPU concentration in this population were not consistent. Although the 325 polymorphism of proCPU has a significant effect on the CPU half-life, its influence on the risk of thrombosis or spontaneous pregnancy loss in carriers of hereditary thrombophilia is not clear. MATERIALS AND METHODS The study population consisted of 144 thrombophilic patients (94 heterozygous and 10 homozygous carriers of FV Leiden, 26 heterozygous carriers of the prothrombin G20210A variation and 14 double carriers of FV Leiden and FII variation) and 69 healthy controls. RESULTS The results show that patients with inherited thrombophilia have a tendency toward lower mean proCPU plasma levels compared to healthy controls, however, this difference was only significant in carriers of FII G20210A (p=0.014). A higher frequency of the most stable Ile325Ile proCPU was seen among carriers of FII G20210A mutation compared to the control group (19% vs 7%; p=0.186). In the second part of the study proCPU as a risk factor for thrombosis was evaluated. In heterozygous carriers of FV Leiden or FII G20210A high levels of proCPU conferred to an almost 4-fold increased risk for spontaneous onset thrombosis. The more stable Ile325Ile proCPU seems to impose a higher risk for clinical manifestation of the thrombophilic condition. Finally, a significant positive correlation between F1+2 and proCPU concentration was seen. CONCLUSION The increased risk of thrombosis in thrombophilia patients is not only ascribable to an increased thrombin generation, but also high levels of proCPU and the presence of the 325Ile genotype tip the balance towards thrombotic tendency even further.

The intrinsic enzymatic activity of procarboxypeptidase U (TAFI) does not significantly influence the fibrinolysis rate: a rebuttal.

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Development of a sensitive and selective assay for the determination of procarboxypeptidase U (thrombin-activatable fibrinolysis inhibitor) in plasma

To date, several assays for procarboxypeptidase U (proCPU) determination exist, all having their own inherent disadvantages and advantages. A drawback of activity-based assays is the interference of the constitutively active carboxypeptidase N (CPN) in plasma. Recent screening of Bz-Xaa-Arg peptides with modified aromatic amino acids at the P1 position revealed a selective CPU substrate, N-benzoyl-ortho-cyano-phenylalanyl-arginine (Bz-o-cyano-Phe-Arg), which will allow straightforward determination of proCPU in plasma. Our assay shows an excellent linearity in the concentration range of 20-2600 U/L, with within- and between-run precision values of 2.7% and 4.6%, respectively. A good correlation with our high-performance liquid chromatography (HPLC)-assisted proCPU activity assay using hippuryl-l-arginine (HipArg) as substrate was found. Besides the major improvement regarding the selectivity, the assay is much easier to perform and far less time-consuming compared with the proCPU activity assay using HipArg as substrate.

Measurement of carboxypeptidase U (active thrombin-activatable fibrinolysis inhibitor) in plasma: Challenges overcome by a novel selective assay.

This article introduces a novel assay for the measurement of carboxypeptidase U (CPU) in plasma using the selective CPU substrate Bz-o-cyano-Phe-Arg (N-benzoyl-ortho-cyano-phenylalanyl-arginine), thereby limiting the interference of plasma carboxypeptidase N (CPN) as well as the intrinsic activity of procarboxypeptidase U (proCPU). A limit of detection of 0.05 U/L (10 pM) was reached. In addition, the current assay has the advantage of being easy to perform and shows excellent linearity and variability, rendering it a useful tool in the screening of samples for the presence of CPU in several patient populations and encouraging in-depth exploration of the pathophysiological role of the proCPU/CPU system.

Comparative study of commercially available procarboxypeptidase U (thrombin-activatable fibrinolysis inhibitor) assays

Carboxypeptidase U (CPU) provides an explicit molecular link between the coagulation and fibrinolytic cascades. By cleaving C-terminal lysines from partially degraded fibrin, CPU prevents the upregulation of plasminogen binding on the fibrin surface, thereby attenuating the fibrinolytic rate [1]. Several studies have been conducted on the proCPU plasma concentration and proCPU genotype as a risk factor for the occurrence of thromboembolic events, and have often led to contradictory conclusions [2]. A possible explanation for these opposing findings could be the fact that a variety of proCPU assays were used, including immunoassays (ELISA) and activity-based assays, many of which are inadequately validated and often appear to be compromised by genotypedependent artefacts [3]. As a result, the numerous published studies describing the biological variation in proCPU and the association of proCPUwith the occurrence of thromboembolic events must be interpreted with caution. In this work, several commercially available proCPU assays were compared and characterized, including the evaluation of the reactivity towards the proCPU 325 polymorphism. With this comparative study, we aimed to emphasize the importance of meticulous characterization of proCPU assays. The results of this study will allow determination of which literature data are obtained with properly validated proCPU assays, and can thus be regarded as reliable. For this study, 132 healthy individuals were included (mean age, 58 ± 17 years). Blood was collected on citrate anticoagulant (109 mM). A fraction of the whole blood was used for genotyping for the proCPU 325 polymorphism, as described previously [4], whereas the remaining fraction was centrifuged for 15 min at 3000 · g. The plasma aliquots were stored at ) 80 C until further analysis. A total of five commercially available proCPU antigen assays were evaluated, i.e. Visulize TAFI (Affinity Biologicals, Ancaster, ON, Canada), Asserachrom TAFI (Diagnostica Stago, Asnières, France), Imuclone TAFI (American Diagnostica, Stamford, CT, USA), Zymutest TAFI (Hyphen BioMed, Neuville-sur-Oise, France), and Zymutest proTAFI (Hyphen BioMed). Additionally, two activity-based commercial proCPU assays were included: Pefakit TAFI (Pentapharm, Basel, Switserland) and Actichrome TAFI (American Diagnostica). The assay procedures were carried out according to the protocols supplied by the manufacturers. For each kit, calibration was performed with a particular reference material supplied individually by the manufacturer. An in-housedeveloped HPLC-assisted assay was also used to measure the proCPU concentration in the samples [5]. Because of the use of a selective CPU substrate, the good precision, and the lack of genotype-dependent artefacts, this in-house-developed assay was used as our reference assay in this comparative study. For the total population (n = 132), ameanproCPUconcentration of 993 ± 161 U/L (mean ± standard deviation [SD]) was found when measured with our reference assay. This concentration is inagreementwithpreviouslypublisheddata for anormalpopulation[6].ThemeanproCPUconcentrationof the studygroupasmeasuredwith the commercial kits corresponded well with the values of a normal population as proposed by the manufacturer(datanotshown).ForActichromeTAFI,however, the proCPU concentrations found were 3.5-fold higher (mean ± SD; 57.5 ± 7.7 lg/mL) than the concentration put forwardintheinsertofthekit(expectedmean:12.0–20.0 lg/mL). Regardless of the assay used, a strong association of the plasma proCPU concentration with the proCPU genotype was found. The proCPU concentration for the homozygous Ile325 individuals was, on average, 11.9% lower than the proCPU plasma concentration in the homozygous Thr325 subgroup when measured with our reference assay. In agreement with these results, around 11% lower proCPU concentrations for the homozygous Ile325 subgroup were found when measured with Asserachrom TAFI, Zymutest proTAFI, Actichrome Correspondence: Dirk F. Hendriks, Laboratory of Medical Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium. Tel: +32 3 265 27 27; fax: +32 3 265 27 45. E-mail: dirk.hendriks@ua.ac.be