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Dirk Hendriks - One of the best experts on this subject based on the ideXlab platform.
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pleiotropic effects of atorvastatin resUlt in a downregUlation of the Carboxypeptidase U system cpU tafia cpb2 in a moUse model of advanced atherosclerosis
Pharmaceutics, 2021Co-Authors: Karen Claesen, Joachim C Mertens, Yani Sim, Lynn Roth, Karlijn Hermans, Dirk HendriksAbstract:Statins (hydroxymethyl-glUtaryl-CoA-redUctase inhibitors) lower proCarboxypeptidase U (proCPU, TAFI, proCPB2). However, it is challenging to prove whether this is a lipid or non-lipid-related pleiotropic effect, since statin treatment decreases cholesterol levels in hUmans. In apolipoprotein E-deficient mice with a heterozygoUs mUtation in the fibrillin-1 gene (ApoE−/−Fbn1C1039G+/−), a model of advanced atherosclerosis, statins do not lower cholesterol. ConseqUently, stUdying cholesterol-independent effects of statins can be achieved more straightforwardly in these mice. Female ApoE −/−Fbn1C1039G+/− mice were fed a Western diet (WD). At week 10 of WD, mice were divided into a WD groUp (receiving WD only) and a WD + atorvastatin groUp (receiving 10 mg/kg/day atorvastatin +WD) groUp. After 15 weeks, blood was collected from the retro-orbital plexUs, and the mice were sacrificed. Total plasma cholesterol and C-reactive protein (CRP) were measUred with commercially available kits. Plasma proCPU levels were determined with an activity-based assay. Total plasma cholesterol levels were not significantly different between both groUps, while proCPU levels were significantly lower in the WD + atorvastatin groUp. Interestingly proCPU levels correlated with CRP and circUlating monocytes. In conclUsion, oUr resUlts confirm that atorvastatin downregUlates proCPU levels in ApoE−/−Fbn1C1039G+/− mice on a WD, and evidence was provided that this downregUlation is a pleiotropic effect of atorvastatin treatment.
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effect of statin therapy on the Carboxypeptidase U cpU tafia cpb2 system in patients with hyperlipidemia a proof of concept observational stUdy
Clinical Therapeutics, 2021Co-Authors: Karen Claesen, Joachim C Mertens, Shahir Basir, Simon De Belder, Jeroen Maes, Johan Bosmans, Hilde Stoffelen, Ingrid De Meester, Dirk HendriksAbstract:ABSTRACT PUrpose Statins are commonly Used in patients with hypercholesterolemia to lower their cholesterol levels and to redUce their cardiovascUlar risk. There is also considerable evidence that statins possess a range of cholesterol-independent effects, inclUding profibrinolytic properties. This pilot stUdy aimed to explore the inflUence of statins on proCarboxypeptidase U (proCPU) biology and to search for possible effects and associations that can be followed Up in a larger stUdy. Methods Blood was collected from 16 patients with hyperlipidemia, before and after 3 months of statin therapy (simvastatin 20 mg or atorvastatin 20 mg). Fifteen age-matched normolipemic persons served as control sUbjects. Lipid parameters and markers of inflammation and fibrinolysis (proCPU levels and clot lysis times) were determined in all samples. Findings Mean (SD) proCPU levels were significantly higher in patients with hypercholesterolemia compared to control sUbjects (1186 [189] U/L vs 1061 [60] U/L). Treatment of these patients with a statin led to a significant average decrease of 11.6% in proCPU levels and broUght the proCPU concentrations to the same level as in the control sUbjects. On a fUnctional level, enhancement in plasma fibrinolytic potential was observed in the statin groUp, with the largest improvement in fibrinolysis seen in patients with the highest baseline proCPU levels and largest proCPU decrease Upon statin treatment. Implications Increased proCPU levels are present in patients with hyperlipidemia. Statin treatment significantly decreased proCPU levels and improved plasma fibrinolysis in these patients. Moreover, oUr stUdy indicates that patients with high baseline proCPU levels are most likely to benefit from statin therapy. The latter shoUld be examined fUrther in a large cohort.
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Carboxypeptidase U cpU tafia cpb2 in thromboembolic disease what do we know three decades after its discovery
International Journal of Molecular Sciences, 2021Co-Authors: Karen Claesen, Joachim C Mertens, Dorien Leenaerts, Dirk HendriksAbstract:ProCarboxypeptidase U (proCPU, TAFI, proCPB2) is a basic Carboxypeptidase zymogen that is converted by thrombin(-thrombomodUlin) or plasmin into the active Carboxypeptidase U (CPU, TAFIa, CPB2), a potent attenUator of fibrinolysis. As CPU forms a molecUlar link between coagUlation and fibrinolysis, the development of CPU inhibitors as profibrinolytic agents constitUtes an attractive new concept to improve endogenoUs fibrinolysis or to increase the efficacy of thrombolytic therapy in thromboembolic diseases. FUrthermore, extensive research has been condUcted on the in vivo role of CPU in (the acUte phase of) thromboembolic disease, as well as on the hypothesis that high proCPU levels and the Thr/Ile325 polymorphism may caUse a thrombotic predisposition. In this paper, an overview is given of the methods available for measUring proCPU, CPU, and inactivated CPU (CPUi), together with a sUmmary of the clinical data generated so far, ranging from the cUrrent knowledge on proCPU concentrations and polymorphisms as potential thromboembolic risk factors to the positioning of different CPU forms (proCPU, CPU, and CPUi) as diagnostic markers for thromboembolic disease, and the potential benefit of pharmacological inhibition of the CPU pathway.
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Carboxypeptidase U cpU Carboxypeptidase b2 activated thrombin activatable fibrinolysis inhibitor inhibition stimUlates the fibrinolytic rate in different in vitro models
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Dorien Leenaerts, Stephane Loyau, Joachim C Mertens, William Boisseau, Jeanbaptiste Michel, Annemarie Lambeir, Martine Jandrotperrus, Dirk HendriksAbstract:Essentials AZD9684 is a potent inhibitor of Carboxypeptidase U (CPU, TAFIa, CPB2). The effect of AZD9684 on fibrinolysis was investigated in foUr in vitro systems. The CPU system also attenUates fibrinolysis in more advanced hemostatic systems. The size of the observed effect on fibrinolysis is dependent on the exact experimental conditions. SUMMARY BackgroUnd Carboxypeptidase U (CPU, Carboxypeptidase B2, activated thrombin-activatable fibrinolysis inhibitor) is a basic Carboxypeptidase that attenUates fibrinolysis. This characteristic has raised interest in the scientific commUnity and pharmaceUtical indUstry for the development of inhibitors as profibrinolytic agents. Objectives Little is known aboUt the contribUtion of CPU to clot resistance in more advanced hemostatic models, which inclUde blood cells and shear stress. The aim of this stUdy was to evalUate the effects of the CPU system in in vitro systems for fibrinolysis with different grades of complexity. Methods The contribUtion of the CPU system was evalUated in the following systems: (i) plasma clot lysis; (ii) rotational thromboelastometry (ROTEM) in whole blood; (iii) front lysis with confocal microscopy in platelet-free and platelet-rich plasma; and (iv) a microflUidic system with whole blood Under arterial shear stress. Experiments were carried oUt in the presence or absence of AZD9684, a specific CPU inhibitor. ResUlts DUring plasma clot lysis, addition of AZD9684 resUlted in 33% faster lysis. In ROTEM, the lysis onset time was decreased by 38%. For both clot lysis and ROTEM, an AZD9684 dose-dependent response was observed. CPU inhibition in front lysis experiments resUlted in 47% and 50% faster lysis for platelet-free plasma and platelet-rich plasma, respectively. Finally, a tendency for faster lysis was observed only in the microflUidic system when AZD9684 was added. ConclUsions Overall, these experiments provide novel evidence that the CPU system can also modUlate fibrinolysis in more advanced hemostatic systems. The extent of the effects appears to be dependent Upon the exact experimental conditions.
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plasma Carboxypeptidase U cpU cpb2 tafia generation dUring in vitro clot lysis and its interplay between coagUlation and fibrinolysis
Thrombosis and Haemostasis, 2017Co-Authors: Dorien Leenaerts, Annemarie Lambeir, Jef Aernouts, Pieter Van Der Veken, Yani Sim, Dirk HendriksAbstract:Carboxypeptidase U (CPU, CPB2, TAFIa) is a basic Carboxypeptidase that is able to attenUate fibrinolysis. The inactive precUrsor proCarboxypeptidase U is converted to its active form by thrombin, the thrombin-thrombomodUlin complex or plasmin. The aim of this stUdy was to investigate and characterise the time coUrse of CPU generation in healthy individUals. In plasma of 29 healthy volUnteers, CPU generation was monitored dUring in vitro clot lysis. CPU activity was measUred by means of an enzymatic assay that Uses the specific sUbstrate Bz-o-cyano-Phe-Arg. An algorithm was written to plot the CPU generation cUrve and calcUlate the parameters that define it. In all individUals, CPU generation was biphasic. Marked inter-individUal differences were present and a reference range was determined. The endogenoUs CPU generation potential is the composite effect of mUltiple factors. With respect to the first CPU activity peak characteristics, we foUnd correlations with baseline proCPU concentration, proCPU Thr325Ile polymorphism, time to clot initiation and the clot lysis time. The second CPU peak related with baseline proCPU levels and with the maximUm tUrbidity of the clot lysis profile. In conclUsion, oUr method offers a techniqUe to determine the endogenoUs CPU generation potential of an individUal. The parameters obtained by the method qUantitatively describe the different mechanisms that inflUence CPU generation dUring the complex interplay between coagUlation and fibrinolysis, which are in line with the threshold hypothesis.
Judith Leurs - One of the best experts on this subject based on the ideXlab platform.
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Carboxypeptidase U tafia a metalloCarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease
Thrombosis and Haemostasis, 2005Co-Authors: Judith Leurs, Dirk HendriksAbstract: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 abil ity 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.
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development of a fast kinetic method for the determination of Carboxypeptidase U tafia Using c terminal arginine containing peptides as sUbstrate
Analytical Biochemistry, 2005Co-Authors: Johan Willemse, Robert Verkerk, Judith Leurs, Dirk HendriksAbstract:Abstract Carboxypeptidase U (CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circUlates in blood as an inactive zymogen, proCarboxypeptidase U, which is activated dUring the process of coagUlation and fibrinolysis. CPU has a very short half-life at 37 °C. Its intrinsic instability complicates the determination of kinetic parameters of different sUbstrates Using an endpoint method. We developed a fast kinetic assay for measUring continUoUsly the release of the C-terminal arginine by CPU independent of the natUre of the sUbstrate peptide Used, allowing Us to perform sUbstrate specificity stUdies of CPU. This method Uses arginine kinase, pyrUvate kinase, and lactate dehydrogenase as aUxiliary enzymes. The CPU activities measUred Using this kinetic assay were in the range of 97–103% of those determined with oUr HPLC-assisted reference assay, and the obtained Km and kcat valUes for hippUryl- l -arginine and bradykinin were in good accordance with those described in the literatUre. As expected, no arginine cleaving was seen Using dipeptides and peptide sUbstrates with a proline in the penUltimate position. The presented kinetic assay enables the fast screening of sUbstrates with a C-terminal arginine and is a valUable new tool for the kinetic evalUation of both synthetic and physiological sUbstrates of CPU.
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development of a fast kinetic method for the determination of Carboxypeptidase U tafia Using c terminal arginine containing peptides as sUbstrate
Analytical Biochemistry, 2005Co-Authors: J L Willemse, Robert Verkerk, Judith Leurs, Dirk HendriksAbstract:Carboxypeptidase U (CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circUlates in blood as an inactive zymogen, proCarboxypeptidase U, which is activated dUring the process of coagUlation and fibrinolysis. CPU has a very short half-life at 37 degrees C. Its intrinsic instability complicates the determination of kinetic parameters of different sUbstrates Using an endpoint method. We developed a fast kinetic assay for measUring continUoUsly the release of the C-terminal arginine by CPU independent of the natUre of the sUbstrate peptide Used, allowing Us to perform sUbstrate specificity stUdies of CPU. This method Uses arginine kinase, pyrUvate kinase, and lactate dehydrogenase as aUxiliary enzymes. The CPU activities measUred Using this kinetic assay were in the range of 97-103% of those determined with oUr HPLC-assisted reference assay, and the obtained K(m) and k(cat) valUes for hippUryl-l-arginine and bradykinin were in good accordance with those described in the literatUre. As expected, no arginine cleaving was seen Using dipeptides and peptide sUbstrates with a proline in the penUltimate position. The presented kinetic assay enables the fast screening of sUbstrates with a C-terminal arginine and is a valUable new tool for the kinetic evalUation of both synthetic and physiological sUbstrates of CPU.
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Carboxypeptidase U tafia prevents lysis from proceeding into the propagation phase throUgh a threshold dependent mechanism
Journal of Thrombosis and Haemostasis, 2004Co-Authors: Judith Leurs, Viveca Nerme, Y Sim, Dirk HendriksAbstract: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.
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different mechanisms contribUte to the biphasic pattern of Carboxypeptidase U tafia generation dUring in vitro clot lysis in hUman plasma
Thrombosis and Haemostasis, 2003Co-Authors: Judith Leurs, Viveca Nerme, Katinka Schatteman, Brittmarie Wissing, Petter Bjorquist, Dirk HendriksAbstract:Carboxypeptidase U (CPU,TAFIa) recently gained interest as a significant player in dampening the fibrinolytic rate. The aim of this stUdy was to investigate the time coUrse of the generation of CPU activity dUring coagUlation and fibrinolysis Using an in vitro clot lysis model in hUman plasma. A first peak of CPU activity appeared after initiation of the coagUlation phase and a second rise in CPU activity was observed dUring the fibrinolysis. The decrease in the proCPU plasma concentration followed the same trend as the appearance of the CPU activity. The direct thrombin inhibitor inogatran eliminated the CPU generation dUring coagUlation bUt not dUring fibrinolysis. Addition of the plasmin inhibitor aprotinin dUring fibrinolysis resUlted in a decrease in CPU activation dUring the lysis phase. These resUlts demonstrate that proCPU was activated dUring coagUlation by thrombin and dUring fibrinolysis by plasmin. Addition of a CPU inhibitor before initiation of clotting decreased the clot lysis time as expected. However, addition in the time period between the two peaks of CPU activity had no apparent effect on the clot lysis time.
Dorien Leenaerts - One of the best experts on this subject based on the ideXlab platform.
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Carboxypeptidase U cpU tafia cpb2 in thromboembolic disease what do we know three decades after its discovery
International Journal of Molecular Sciences, 2021Co-Authors: Karen Claesen, Joachim C Mertens, Dorien Leenaerts, Dirk HendriksAbstract:ProCarboxypeptidase U (proCPU, TAFI, proCPB2) is a basic Carboxypeptidase zymogen that is converted by thrombin(-thrombomodUlin) or plasmin into the active Carboxypeptidase U (CPU, TAFIa, CPB2), a potent attenUator of fibrinolysis. As CPU forms a molecUlar link between coagUlation and fibrinolysis, the development of CPU inhibitors as profibrinolytic agents constitUtes an attractive new concept to improve endogenoUs fibrinolysis or to increase the efficacy of thrombolytic therapy in thromboembolic diseases. FUrthermore, extensive research has been condUcted on the in vivo role of CPU in (the acUte phase of) thromboembolic disease, as well as on the hypothesis that high proCPU levels and the Thr/Ile325 polymorphism may caUse a thrombotic predisposition. In this paper, an overview is given of the methods available for measUring proCPU, CPU, and inactivated CPU (CPUi), together with a sUmmary of the clinical data generated so far, ranging from the cUrrent knowledge on proCPU concentrations and polymorphisms as potential thromboembolic risk factors to the positioning of different CPU forms (proCPU, CPU, and CPUi) as diagnostic markers for thromboembolic disease, and the potential benefit of pharmacological inhibition of the CPU pathway.
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selective inhibition of Carboxypeptidase U may redUce microvascUlar thrombosis in rat experimental stroke
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Joachim C Mertens, Dorien Leenaerts, Stephane Loyau, William Boisseau, Jeanbaptiste Michel, Annemarie Lambeir, Martine Jandrotperrus, Lucas Di Meglio, Celina Ducroux, Mikael MazighiAbstract:BackgroUnd Carboxypeptidase U (CPU, CPB2, TAFIa) is a potent attenUator of fibrinolysis. The inhibition of CPU is thUs an interesting strategy for improving thrombolysis. Objectives The time coUrse of CPU generation and proCPU consUmption were assessed in an experimental rat model of acUte ischemic stroke (AIS). In addition, the effects of the selective CPU inhibitor AZD9684 on CPU kinetics, microvascUlar thrombosis (MT), and AIS oUtcome were evalUated. Methods Rats were sUbjected to transient middle cerebral artery occlUsion (tMCAO) and received recombinant tissUe-type plasminogen activator (tPA), a specific CPU inhibitor (AZD9684), combination therapy of tPA and AZD9684, or saline for 1 hoUr Using a randomized treatment regime. CPU and proCPU levels were determined at five time points and assessed in light of oUtcome parameters (a.o.: infarct volUme and fibrin[ogen] deposition as a measUre for MT). ResUlts Clear activation of the CPU system was observed after AIS indUction, in both saline- and tPA-treated rats. Maximal CPU activities were observed at treatment cessation and were higher in tPA-treated animals compared to the saline groUp. Concomitant proCPU consUmption was more pronoUnced in tPA-treated rats. AZD9684 sUppressed the CPU activity and redUced fibrin(ogen) deposition, sUggesting a redUction of MT. Nonetheless, a significant decrease in infarct volUme was not observed. ConclUsions A pronoUnced activation of the CPU system was observed dUring tMCAO in rats. Selective inhibition of CPU with AZD9684 was able to redUce fibrin(ogen) deposition and brain edema, sUggesting a redUction of MT bUt withoUt a significant effect on final infarct volUme.
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Carboxypeptidase U cpU Carboxypeptidase b2 activated thrombin activatable fibrinolysis inhibitor inhibition stimUlates the fibrinolytic rate in different in vitro models
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Dorien Leenaerts, Stephane Loyau, Joachim C Mertens, William Boisseau, Jeanbaptiste Michel, Annemarie Lambeir, Martine Jandrotperrus, Dirk HendriksAbstract:Essentials AZD9684 is a potent inhibitor of Carboxypeptidase U (CPU, TAFIa, CPB2). The effect of AZD9684 on fibrinolysis was investigated in foUr in vitro systems. The CPU system also attenUates fibrinolysis in more advanced hemostatic systems. The size of the observed effect on fibrinolysis is dependent on the exact experimental conditions. SUMMARY BackgroUnd Carboxypeptidase U (CPU, Carboxypeptidase B2, activated thrombin-activatable fibrinolysis inhibitor) is a basic Carboxypeptidase that attenUates fibrinolysis. This characteristic has raised interest in the scientific commUnity and pharmaceUtical indUstry for the development of inhibitors as profibrinolytic agents. Objectives Little is known aboUt the contribUtion of CPU to clot resistance in more advanced hemostatic models, which inclUde blood cells and shear stress. The aim of this stUdy was to evalUate the effects of the CPU system in in vitro systems for fibrinolysis with different grades of complexity. Methods The contribUtion of the CPU system was evalUated in the following systems: (i) plasma clot lysis; (ii) rotational thromboelastometry (ROTEM) in whole blood; (iii) front lysis with confocal microscopy in platelet-free and platelet-rich plasma; and (iv) a microflUidic system with whole blood Under arterial shear stress. Experiments were carried oUt in the presence or absence of AZD9684, a specific CPU inhibitor. ResUlts DUring plasma clot lysis, addition of AZD9684 resUlted in 33% faster lysis. In ROTEM, the lysis onset time was decreased by 38%. For both clot lysis and ROTEM, an AZD9684 dose-dependent response was observed. CPU inhibition in front lysis experiments resUlted in 47% and 50% faster lysis for platelet-free plasma and platelet-rich plasma, respectively. Finally, a tendency for faster lysis was observed only in the microflUidic system when AZD9684 was added. ConclUsions Overall, these experiments provide novel evidence that the CPU system can also modUlate fibrinolysis in more advanced hemostatic systems. The extent of the effects appears to be dependent Upon the exact experimental conditions.
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plasma Carboxypeptidase U cpU cpb2 tafia generation dUring in vitro clot lysis and its interplay between coagUlation and fibrinolysis
Thrombosis and Haemostasis, 2017Co-Authors: Dorien Leenaerts, Annemarie Lambeir, Jef Aernouts, Pieter Van Der Veken, Yani Sim, Dirk HendriksAbstract:Carboxypeptidase U (CPU, CPB2, TAFIa) is a basic Carboxypeptidase that is able to attenUate fibrinolysis. The inactive precUrsor proCarboxypeptidase U is converted to its active form by thrombin, the thrombin-thrombomodUlin complex or plasmin. The aim of this stUdy was to investigate and characterise the time coUrse of CPU generation in healthy individUals. In plasma of 29 healthy volUnteers, CPU generation was monitored dUring in vitro clot lysis. CPU activity was measUred by means of an enzymatic assay that Uses the specific sUbstrate Bz-o-cyano-Phe-Arg. An algorithm was written to plot the CPU generation cUrve and calcUlate the parameters that define it. In all individUals, CPU generation was biphasic. Marked inter-individUal differences were present and a reference range was determined. The endogenoUs CPU generation potential is the composite effect of mUltiple factors. With respect to the first CPU activity peak characteristics, we foUnd correlations with baseline proCPU concentration, proCPU Thr325Ile polymorphism, time to clot initiation and the clot lysis time. The second CPU peak related with baseline proCPU levels and with the maximUm tUrbidity of the clot lysis profile. In conclUsion, oUr method offers a techniqUe to determine the endogenoUs CPU generation potential of an individUal. The parameters obtained by the method qUantitatively describe the different mechanisms that inflUence CPU generation dUring the complex interplay between coagUlation and fibrinolysis, which are in line with the threshold hypothesis.
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plasma levels of Carboxypeptidase U cpU cpb2 or tafia are elevated in patients with acUte myocardial infarction
Journal of Thrombosis and Haemostasis, 2015Co-Authors: Dorien Leenaerts, Annemarie Lambeir, Yani Sim, J M Bosmans, P Van Der Veken, Dirk HendriksAbstract:SUmmary BackgroUnd Two decades after its discovery, Carboxypeptidase U (CPU, CPB2 or TAFIa) has become a compelling drUg target in thrombosis research. However, given the difficUlty of measUring CPU in the blood circUlation and the demanding sample collecton reqUirements, previoUs clinical stUdies focUsed mainly on measUring its inactive precUrsor, proCPU (proCPB2 or TAFI). Objectives Using a sensitive and specific enzymatic assay, we investigated plasma CPU levels in patients presenting with acUte myocardial infarction (AMI) and in controls. Methods In this case–control stUdy, peripheral arterial blood samples were collected from 45 patients with AMI (25 with ST segment elevation myocardial infarction [STEMI], 20 with non-ST segment elevation myocardial infarction [NSTEMI]) and 42 controls. Additionally, intracoronary blood samples were collected from 11 STEMI patients dUring thrombUs aspiration. SUbseqUently, proCPU and CPU plasma concentrations in all samples were measUred by means of an activity-based assay, Using Bz-o-cyano-Phe-Arg as a selective sUbstrate. ResUlts CPU activity levels were higher in patients with AMI (median LOD-LOQ, range 0–1277 mU L−1) than in controls (median < LOD, range 0–128 mU L−1). No correlation was foUnd between CPU levels and AMI type (NSTEMI [median between LOD-LOQ, range 0–465 mU L−1] vs. STEMI [median between LOD-LOQ, range 0–1277 mU L−1]). Intracoronary samples (median 109 mU L−1, range 0–759 mU L−1) contained higher CPU levels than did peripheral samples (median between LOD-LOQ, range 0–107 mU L−1), indicating increased local CPU generation. With regard to proCPU, we foUnd lower levels in AMI patients (median 910 U L−1, range 706–1224 U L−1) than in controls (median 1010 U L−1, range 753–1396 U L−1). ConclUsions AMI patients have higher plasma CPU levels and lower proCPU levels than controls. This finding indicates in vivo generation of fUnctional active CPU in patients with AMI.
Evelien Heylen - One of the best experts on this subject based on the ideXlab platform.
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an Update on the role of Carboxypeptidase U tafia in fibrinolysis
Frontiers in Bioscience, 2011Co-Authors: Evelien Heylen, J L Willemse, Dirk HendriksAbstract: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.
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measUrement of Carboxypeptidase U active thrombin activatable fibrinolysis inhibitor in plasma challenges overcome by a novel selective assay
Analytical Biochemistry, 2010Co-Authors: Evelien Heylen, Sebastiaan Van Goethem, Koen Augustyns, Dirk HendriksAbstract: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.
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Carboxypeptidase U tafia a new drUg target for fibrinolytic therapy
Journal of Thrombosis and Haemostasis, 2009Co-Authors: J L Willemse, Evelien Heylen, Michael E Nesheim, Dirk HendriksAbstract: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.
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Carboxypeptidase U tafia decreases the efficacy of thrombolytic therapy in ischemic stroke patients
Clinical Neurology and Neurosurgery, 2009Co-Authors: Raf Brouns, Dirk Hendriks, J L Willemse, Evelien Heylen, Rishi Sheorajpanday, Jan Kunnen, Didier De Surgeloose, Peter Paul De DeynAbstract:Abstract 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.
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Inhibition of Carboxypeptidase U (TAFIa) activity improves rt-PA indUced thrombolysis in a dog model of coronary artery thrombosis.
Thrombosis research, 2005Co-Authors: Jan-arne Björkman, Tommy Abrahamsson, Viveca Nerme, Christer MattssonAbstract:The objective of this stUdy was to test the hypothesis if thrombolysis indUced by recombinant tissUe-type plasminogen activator, (rt-PA) coUld be facilitated by inhibiting Carboxypeptidase U (CPU, active Thrombin Activatable Fibrinolysis Inhibitor, TAFIa) activity. The efficacy of rt-PA alone, or in combination with the Carboxypeptidase inhibitor MERGETPA, was compared in a dog model of coronary artery thrombosis. Twenty dogs were randomised in two groUps, one received rt-PA, 1 mg kg(-1), as intravenoUs infUsion over 20 min starting 30 min after thrombUs formation, and the other groUp received rt-PA, 1 mg kg(-1), as groUp one with the addition of MERGEPTA 5 mg kg(-1) starting 25 min prior to coronary artery occlUsion and followed by infUsion of 5 mg kg(-1) h(-1) Until the end of experiment. Efficacy was assessed by determination of time to lysis, dUration of patency and blood flow dUring patency. Both groUps had similar baseline characteristics with respect to haemodynamic parameters, i.e., heart rate, blood pressUre and coronary artery blood flow. Coadministration of rt-PA and MERGETPA resUlted in significant decrease in time to lysis (15+/-1.5 min vs. 20+/-1.7 min, p=0.03), increased patency time (87+/-16 min vs. 46+/-12 min, p=0.047) and increased coronary blood flow dUring patency (1131 mL h(-1) vs. 405 mL h(-1), p=0.015), compared to rt-PA alone. These resUlts indicate that an inhibitor of CPU activity may have a beneficial effect in patients Undergoing thrombolytic therapy by attaining shorter time to reperfUsion and improved coronary patency.
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Local proCPU (TAFI) activation dUring thrombolytic treatment in a dog model of coronary artery thrombosis can be inhibited with a direct, small molecUle thrombin inhibitor (melagatran).
Thrombosis and haemostasis, 2002Co-Authors: Christer Mattsson, Simon Scharpé, Viveca Nerme, J. A. Björkman, T. Abrahamsson, Katinka Schatteman, Judith Leurs, Dirk HendriksAbstract:To test the hypothesis that the direct thrombin inhibitor, melagatran is able to inhibit local pro-Carboxypeptidase U (proCPU) activation that occUrs dUring thrombolytic treatment, t-PA alone, or in combination with melagatran, was given to dogs with a coronary artery thrombosis. Blood samples from the great cardiac vein and aorta were collected at baseline, dUring thrombUs formation, throUghoUt the t-PA±melagatran infUsion and dUring the patency period, for analysis of CPU activity Using a novel assay. A higher CPU activity in venoUs compared to arterial blood (V-A difference) indicates CPU activation in coronary vessels. Efficacy was assessed by determination of time to lysis, dUration of patency and blood flow dUring patency. Dogs (n = 26) were randomized to receive either 1) t-PA, 1 mg/kg as an intravenoUs 20-min infUsion; 2) t-PA as in groUp 1,+ melagatran bolUs, 0.3 mg/kg, followed by a 3-h infUsion (0.15 mg/kg per h); 3) sham-operated bUt no coronary thrombUs, and administered t-PA as for GroUp 1. All groUps had similar baseline characteristics. Significant increases in CPU activity were observed in GroUps 1 and 2 dUring thrombUs formation, with V-A differences of 5.5 and 4.5 U/L, respectively. No significant V-A difference was observed in the sham-operated groUp. CPU activity increased in GroUp 1 dUring the t-PA infUsion (V-A difference 15.9 U/L), whereas the V-A difference in GroUp 2 decreased to 2.6 U/L following melagatran treatment. These resUlts demonstrate that melagatran attenUates generation of CPU in the coronary circUlation. The mechanism is probably indirect, via inhibition of thrombin-mediated activation of proCPU.