Johan Willemse

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.

Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.

Procarboxypeptidase U [proCPU, thrombin‐activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down‐regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPUs activity. We have shown that single as well as a few 2–4 mutations in human CPU can prolong the half‐life of CPUs activity at 37 °C from 0.2 h of wild‐type CPU to 0.5–5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis.

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.

Influence of separator gel in Sarstedt S-Monovette® serum tubes on various therapeutic drugs, hormones, and proteins.

BACKGROUND A separator or barrier gel is a common component of serum and plasma collection tubes. Despite their advantages, the use of these tubes is not universally accepted, especially for therapeutic drug monitoring (TDM). The aim of this study was to evaluate whether the polyacrylester separator gel in Sarstedt S-Monovette\® tubes influences the concentration of 10 selected parameters (amikacin, vancomycin, valproic acid, acetaminophen, cortisol, free thyroxine, thyroid-stimulating hormone, transferrin, prealbumin and carcinoembryonic antigen) in a clinically significant way. METHODS Results from patient samples collected in plastic Sarstedt S-Monovette® tubes with separator gel were compared with those from plain serum sample tubes. Analytes were measured in both tubes on 4 consecutive days to study the influence of prolonged contact with the separator gel. Between analyses tubes were stored at 4°C. Stability was also evaluated over 72 h for each collection tube. When statistical differences were detected, the clinical significance was evaluated based on the total allowable error (TEa). RESULTS On day 1 no statistically significant differences were observed between samples collected in Sarstedt S-Monovette® tubes with and without separator gel. Statistical differences were present from day 2 on, but were not clinically significant. All evaluated parameters were clinically stable over 72 h at 4°C based on TEa, except for transferrin en fT4. CONCLUSION The separator gel in Sarstedt S-Monovette® tubes did not show statistically significant differences on the day of phlebotomy. Later on statistically significant differences appeared but except for the stability of fT4 and transferrin they all remained clinically insignificant.

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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Discovery of thrombin activatable fibrinolysis inhibitor (TAFI)

CAS: blood clotting factor 11, 9013-55-2; thrombin, 9002-04-4; tissue plasminogen activator, 105913-11-9; protein C, 60202-16-6; Carboxypeptidase U, 3.4.17.20; Protein C; Tissue Plasminogen Activator, 3.4.21.68