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1.
J Thromb Haemost ; 21(5): 1266-1273, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36740042

RESUMO

BACKGROUND: Statins efficiently lower cholesterol and also exert pleiotropic effects that extend beyond lipid lowering. In a recent pilot study, valuable information on the carboxypeptidase U (CPU) system in hyperlipidemia and the effect of statin therapy was collected. It was shown that proCPU levels are increased in hyperlipidemic patients. Statins significantly decreased proCPU levels and improved plasma fibrinolysis. Furthermore, it was suggested that patients with high baseline proCPU levels are most likely to benefit from statin therapy. OBJECTIVES: We aimed to further substantiate the effect of hyperlipidemia and statin therapy on CPU-related parameters in a larger cohort of hyperlipidemic and statin-treated individuals. METHODS: Blood was collected from 141 individuals treated with different dosages of atorvastatin (10-80 mg), 38 normolipidemic, and 37 hyperlipidemic controls. Lipid parameters and markers of fibrinolysis (proCPU and clot lysis time) were determined and compared between the groups. RESULTS: Pilot study results of high proCPU concentrations in hyperlipidemic patients and the proCPU-reducing effect of atorvastatin were confirmed. Accordingly, an improvement in plasma fibrinolytic potential was seen under the influence of atorvastatin. High interindividual variation in proCPU concentrations was observed in the hyperlipidemic cohort, with up to 80% higher proCPU levels compared with normolipidemic controls. Furthermore, proCPU concentration and the dosage of atorvastatin were inversely correlated. CONCLUSIONS: This study clearly shows that plasma proCPU concentrations and its expected effect on the fibrinolytic rate (as measured by clot lysis time) are increased in hyperlipidemic patients and that these effects can be normalized (and even further reduced compared with normolipidemic patients) by atorvastatin treatment.


Assuntos
Carboxipeptidase B2 , Inibidores de Hidroximetilglutaril-CoA Redutases , Humanos , Atorvastatina/farmacologia , Atorvastatina/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Projetos Piloto , Terapia Trombolítica
2.
Int J Mol Sci ; 24(4)2023 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-36835137

RESUMO

Carboxypeptidase U (CPU, TAFIa, CPB2) is a potent attenuator of fibrinolysis that is mainly synthesized by the liver as its inactive precursor proCPU. Aside from its antifibrinolytic properties, evidence exists that CPU can modulate inflammation, thereby regulating communication between coagulation and inflammation. Monocytes and macrophages play a central role in inflammation and interact with coagulation mechanisms resulting in thrombus formation. The involvement of CPU and monocytes/macrophages in inflammation and thrombus formation, and a recent hypothesis that proCPU is expressed in monocytes/macrophages, prompted us to investigate human monocytes and macrophages as a potential source of proCPU. CPB2 mRNA expression and the presence of proCPU/CPU protein were studied in THP-1, PMA-stimulated THP-1 cells and primary human monocytes, M-CSF-, IFN-γ/LPS-, and IL-4-stimulated-macrophages by RT-qPCR, Western blotting, enzyme activity measurements, and immunocytochemistry. CPB2 mRNA and proCPU protein were detected in THP-1 and PMA-stimulated THP-1 cells as well as in primary monocytes and macrophages. Moreover, CPU was detected in the cell medium of all investigated cell types and it was demonstrated that proCPU can be activated into functionally active CPU in the in vitro cell culture environment. Comparison of CPB2 mRNA expression and proCPU concentrations in the cell medium between the different cell types provided evidence that CPB2 mRNA expression and proCPU secretion in monocytes and macrophages is related to the degree to which these cells are differentiated. Our results indicate that primary monocytes and macrophages express proCPU. This sheds new light on monocytes and macrophages as local proCPU sources.


Assuntos
Carboxipeptidase B2 , Macrófagos , Monócitos , Humanos , Carboxipeptidase B2/genética , Carboxipeptidase B2/metabolismo , Diferenciação Celular/genética , Inflamação , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Monócitos/metabolismo , RNA Mensageiro
3.
J Clin Med ; 11(6)2022 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-35329820

RESUMO

Coronavirus disease 2019 (COVID-19) is a viral lower respiratory tract infection caused by the highly transmissible and pathogenic SARS-CoV-2 (severe acute respiratory-syndrome coronavirus-2). Besides respiratory failure, systemic thromboembolic complications are frequent in COVID-19 patients and suggested to be the result of a dysregulation of the hemostatic balance. Although several markers of coagulation and fibrinolysis have been studied extensively, little is known about the effect of SARS-CoV-2 infection on the potent antifibrinolytic enzyme carboxypeptidase U (CPU). Blood was collected longitudinally from 56 hospitalized COVID-19 patients and 32 healthy controls. Procarboxypeptidase U (proCPU) levels and total active and inactivated CPU (CPU+CPUi) antigen levels were measured. At study inclusion (shortly after hospital admission), proCPU levels were significantly lower and CPU+CPUi antigen levels significantly higher in COVID-19 patients compared to controls. Both proCPU and CPU+CPUi antigen levels showed a subsequent progressive increase in these patients. Hereafter, proCPU levels decreased and patients were, at discharge, comparable to the controls. CPU+CPUi antigen levels at discharge were still higher compared to controls. Baseline CPU+CPUi antigen levels (shortly after hospital admission) correlated with disease severity and the duration of hospitalization. In conclusion, CPU generation with concomitant proCPU consumption during early SARS-CoV-2 infection will (at least partly) contribute to the hypofibrinolytic state observed in COVID-19 patients, thus enlarging their risk for thrombosis. Moreover, given the association between CPU+CPUi antigen levels and both disease severity and duration of hospitalization, this parameter may be a potential biomarker with prognostic value in SARS-CoV-2 infection.

4.
Clin Chim Acta ; 531: 4-11, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35283094

RESUMO

BACKGROUND: COVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospitalized COVID-19 patients. METHODS: Fifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longitudinal follow-up samples. RESULTS: Compared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 patients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge. CONCLUSION: The changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel.


Assuntos
COVID-19 , Choque Séptico , Carboxipeptidases , Dipeptidil Peptidase 4 , Endopeptidases , Gelatinases , Humanos , Proteínas de Membrana , Peptídeo Hidrolases , Prolina , Serina Endopeptidases
5.
Transl Stroke Res ; 13(6): 959-969, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-34796454

RESUMO

The antifibrinolytic enzyme carboxypeptidase U (CPU, TAFIa, CPB2) is an appealing target for the treatment of acute ischemic stroke (AIS). Increased insights in CPU activation and inactivation during thrombolysis (rtPA) with or without endovascular thrombectomy (EVT) are required to develop CPU inhibitors as profibrinolytic agents with optimal benefits/risks. Therefore, CPU kinetics during ischemic stroke treatment were evaluated. AIS patients with documented cerebral artery occlusion receiving rtPA (N = 20) or rtPA + EVT (N = 16) were included. CPU activation during thrombolysis was measured by an ultrasensitive HPLC-based CPU activity method and by an ELISA measuring both CPU and inactivated CPU (CPU + CPUi). Intravenous blood samples were collected at admission and throughout the first 24 h. Additional in situ blood samples were collected in the rtPA + EVT cohort proximal from the thrombus. The NIHSS score was determined at baseline and 24 h. CPU activity and CPU + CPUi levels increased upon rtPA administration and reached peak values at the end of thrombolysis (1 h). High inter-individual variability was observed in both groups. CPU activity decreased rapidly within 3 h, while CPU + CPUi levels were still elevated at 7 h. CPU activity or CPU + CPUi levels were similar in in situ and peripheral samples. No correlation between CPU or CPU + CPUi and NIHSS or thrombus localization was found. The CPU system was rapidly activated and deactivated following thrombolysis and thrombectomy in stroke patients, suggesting that a CPU inhibitor would have to be administered during rtPA infusion and over the next few hours. The high CPU generation variability suggests that some patients may not respond to the treatment. EudraCT number 2017-002760-41.


Assuntos
Carboxipeptidase B2 , AVC Isquêmico , Acidente Vascular Cerebral , Trombose , Humanos , Carboxipeptidase B2/fisiologia , Trombectomia , Ativador de Plasminogênio Tecidual/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/cirurgia , Terapia Trombolítica/métodos , Resultado do Tratamento
6.
Pharmaceutics ; 13(10)2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34684024

RESUMO

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.

7.
Clin Ther ; 43(5): 908-916, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33910760

RESUMO

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.


Assuntos
Carboxipeptidase B2 , Inibidores de Hidroximetilglutaril-CoA Redutases , Hiperlipidemias , Fibrinólise , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Projetos Piloto
8.
Int J Mol Sci ; 22(2)2021 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-33477318

RESUMO

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.


Assuntos
Carboxipeptidase B2/metabolismo , Carboxipeptidase B2/fisiologia , Tromboembolia/metabolismo , Coagulação Sanguínea/fisiologia , Carboxipeptidase B2/genética , Fibrinolisina/metabolismo , Fibrinólise/fisiologia , Genótipo , Humanos , Trombina/metabolismo , Tromboembolia/fisiopatologia , Terapia Trombolítica/métodos , Trombose/metabolismo
9.
J Thromb Haemost ; 18(12): 3325-3335, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32869423

RESUMO

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.


Assuntos
Isquemia Encefálica , Carboxipeptidase B2 , Acidente Vascular Cerebral , Trombose , Animais , Fibrinólise , Ratos , Acidente Vascular Cerebral/tratamento farmacológico , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tecidual
10.
J Thromb Haemost ; 18(9): 2209-2214, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32634856

RESUMO

BACKGROUND: Thrombomodulin-associated coagulopathy (TM-AC) is a rare bleeding disorder in which a single reported p.Cys537* variant in the thrombomodulin gene THBD causes high plasma thrombomodulin (TM) levels. High TM levels attenuate thrombin generation and delay fibrinolysis. OBJECTIVES: To report the characteristics of pedigree with a novel THBD variant causing TM-AC, and co-inherited deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI). PATIENTS/METHODS: Identification of pathogenic variants in hemostasis genes by next-generation sequencing and case recall for deep phenotyping. RESULTS: Pedigree members with a previously reported THBD variant predicting p.Pro496Argfs*10 and chain truncation in TM transmembrane domain had abnormal bleeding and greatly increased plasma TM levels. Affected cases had attenuated thrombin generation and delayed fibrinolysis similar to previous reported TM_AC cases with THBD p.Cys537*. Coincidentally, some pedigree members also harbored a stop-gain variant in CPB2 encoding TAFI. This reduced plasma TAFI levels but was asymptomatic. Pedigree members with TM-AC caused by the p.Pro496Argfs*10 THBD variant and also TAFI deficiency had a partially attenuated delay in fibrinolysis, but no change in the defective thrombin generation. CONCLUSIONS: These data extend the reported genetic repertoire of TM-AC and establish a common molecular pathogenesis arising from high plasma levels of TM extra-cellular domain. The data further confirm that the delay in fibrinolysis associated with TM-AC is directly linked to increased TAFI activation. The combination of the rare variants in the pedigree members provides a unique genetic model to develop understanding of the thrombin-TM system and its regulation of TAFI.


Assuntos
Transtornos da Coagulação Sanguínea , Carboxipeptidase B2 , Carboxipeptidase B2/genética , Fibrinólise/genética , Humanos , Linhagem , Trombina , Trombomodulina/genética
11.
J Thromb Haemost ; 17(6): 878-884, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30887647

RESUMO

Essentials Hemolytic influence on the (pro)carboxypeptidase U ((pro)CPU) system is not known. In the current manuscript, this was assessed by spiking pooled normal plasma with hemolysate. CPU activity, proCPU levels, and clot lysis times showed a dose-dependent hemolytic bias. The observed bias in the several CPU related parameters is due to inhibition of CPU activity. INTRODUCTION: Spurious hemolysis of samples is the leading cause of interference in coagulation testing and was described to interfere in fibrinolysis assays. The influence of hemolysis on the procarboxypeptidase U (proCPU) system is not known. METHODS: By means of spiking of hemolysate in pooled normal plasma, the effect of hemolysis on CPU, proCPU, and functional clot lysis assays was assessed. The influence of hemolysis on CPU generation during in vitro clot lysis was also evaluated. Cutoffs corresponding to maximal acceptable bias were determined. RESULTS AND DISCUSSION: When active CPU was added to pooled plasma, a severe decrease in activity - up to 97.2% inhibition - was seen with increasing plasma concentrations of oxyhemoglobin (oxyHb) and the 10% cutoff value was found to be 0.3 g/L oxyHb. Using an activity-based assay, proCPU levels appeared to decrease gradually with increased hemolysis (maximal reduction of 19.5%) with a 10% cutoff value of 4.2 g/L oxyHb. The relative clot lysis time (CLT) showed a maximal negative bias of 68.5%. The reduction in CLT paralleled a significant reduction of the first CPU activity peak during clot lysis. The cutoff value for the CLT was 0.4 g/L oxyHb. In presence of thrombomodulin (TM), CLT+TM was not affected up to 8.0 g/L oxyHb. CONCLUSION: These data indicate a clear inhibition of the CPU system because of hemolysis resulting in an increase of lysis in functional fibrinolysis assays. We were able to quantify the inhibitory effect and to propose cutoff values for every parameter.


Assuntos
Testes de Coagulação Sanguínea/métodos , Carboxipeptidase B2/antagonistas & inibidores , Carboxipeptidase B2/sangue , Hemólise/fisiologia , Testes de Coagulação Sanguínea/estatística & dados numéricos , Tempo de Lise do Coágulo de Fibrina/métodos , Tempo de Lise do Coágulo de Fibrina/estatística & dados numéricos , Fibrinólise/fisiologia , Voluntários Saudáveis , Humanos , Técnicas In Vitro
12.
Thromb Haemost ; 117(8): 1498-1508, 2017 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-28692110

RESUMO

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.


Assuntos
Coagulação Sanguínea , Carboxipeptidase B2/sangue , Fibrinólise , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Biomarcadores/sangue , Coagulação Sanguínea/genética , Testes de Coagulação Sanguínea , Carboxipeptidase B2/genética , Ativação Enzimática , Feminino , Fibrinolisina/metabolismo , Fibrinólise/genética , Genótipo , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo Genético , Trombina/metabolismo , Trombomodulina/sangue , Fatores de Tempo , Adulto Jovem
13.
Transl Stroke Res ; 8(2): 157-164, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27561653

RESUMO

Dipeptidyl peptidase IV (DPPIV) inhibition may be a promising therapeutic strategy for acute stroke treatment, given its potential to prolong the biological half-life of neuroprotective substrates. A related protease, fibroblast activation protein (FAP), was recently shown to inactivate the same substrates. Therefore, it should also be investigated as a potential target in stroke. The study aimed to investigate whether stroke severity and outcome correlate with DPPIV and FAP activities and their kinetics shortly after acute ischemic stroke. DPPIV and FAP activities were analyzed in the serum of 50 hyperacute stroke patients at admission, 1 day, 3 days, and 7 days after stroke onset and in 50 age-matched healthy controls. This was done as part of the Middelheim's Interdisciplinary Stroke Study. DPPIV activity tended to increase shortly after stroke compared to the control population. DPPIV and FAP activities steadily decreased in the first week after stroke onset. Higher infarct volumes (≥5 ml) and a more severe stroke (NIHSS >7) at admission were correlated with a stronger decrease in the activities of both enzymes. Moreover, these patients more often developed a progressive stroke, were more often institutionalized. Patients with a stronger increase in DPPIV activity at admission and decrease in the activity of both DPPIV and FAP during the first week after stroke onset had a more severe stroke and worse short-term outcomes.


Assuntos
Isquemia Encefálica/enzimologia , Dipeptidil Peptidase 4/sangue , Gelatinases/sangue , Proteínas de Membrana/sangue , Serina Endopeptidases/sangue , Acidente Vascular Cerebral/enzimologia , Idoso , Isquemia Encefálica/sangue , Isquemia Encefálica/epidemiologia , Progressão da Doença , Endopeptidases , Feminino , Humanos , Masculino , Índice de Gravidade de Doença , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/epidemiologia
14.
Neurochem Res ; 40(1): 81-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25370794

RESUMO

Prolyl carboxypeptidase (PRCP) is an enzyme associated with cerebrovascular risk factors such as hypertension, diabetes mellitus, obesity and hyperlipidemia. We aim to evaluate the relation between serum PRCP activity and severity, evolution and outcome of acute ischemic stroke. We used a specific RP-HPLC activity assay to measure PRCP activity in serum of 50 stroke patients at admission, and at 24 h, 72 h and 7 days after stroke onset to assess correlations with stroke severity based on the National Institutes of Health Stroke scale score (NIHSS), infarct volume on brain MRI scan, stroke outcome based on the modified Rankin scale (mRS) and mortality at 3 months after stroke. The average PRCP activity in serum decreased significantly the first 24 h after stroke onset and returned to baseline values at day 7. High NIHSS scores and infarct volumes at admission were related with a more pronounced decrease of PRCP in the first 24 h after stroke (ΔPRCP24, r = 0.31, P < 0.05; r = 0.30, P < 0.05). In addition, patients who displayed a more pronounced decrease in PRCP levels during the first 24 h after stroke were more likely to be institutionalized upon discharge (n = 21) (ΔPRCP24 ± SD, 0.05 ± 0.10 U/L vs. 0.17 ± 0.14 U/L, P = 0.001). The decrease in PRCP levels in the first 24 h after stroke onset is associated with stroke severity and an unfavourable short-term stroke outcome.


Assuntos
Isquemia Encefálica/enzimologia , Carboxipeptidases/metabolismo , Acidente Vascular Cerebral/enzimologia , Idoso , Isquemia Encefálica/patologia , Carboxipeptidases/sangue , Progressão da Doença , Feminino , Humanos , Masculino , Fatores de Risco , Acidente Vascular Cerebral/patologia , Resultado do Tratamento
15.
J Histochem Cytochem ; 61(3): 218-35, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23172796

RESUMO

Although the kidney generally has been regarded as an excellent source of carboxypeptidase M (CPM), little is known about its renal-specific expression level and distribution. This study provides a detailed localization of CPM in healthy and diseased human kidneys. The results indicate a broad distribution of CPM along the renal tubular structures in the healthy kidney. CPM was identified at the parietal epithelium beneath the Bowman's basement membrane and in glomerular mesangial cells. Capillaries, podocytes, and most interstitial cells were CPM negative. Tumor cells of renal cell carcinoma subtypes lose CPM expression upon dedifferentiation. Tissue microarray analysis demonstrated a correlation between low CPM expression and tumor cell type. CPM staining was intense on phagocytotic tumor-associated macrophages. Immunoreactive CPM was also detected in the tumor-associated vasculature. The absence of CPM in normal renal blood vessels points toward a role for CPM in angiogenesis. Coexistence of CPM and the epidermal growth factor receptor (EGFR) was detected in papillary renal cell carcinoma. However, the different subcellular localization of CPM and EGFR argues against an interaction between these h proteins. The description of the distribution of CPM in human kidney forms the foundation for further study of the (patho)physiological activities of CPM in the kidney.


Assuntos
Carcinoma de Células Renais/enzimologia , Neoplasias Renais/enzimologia , Rim/enzimologia , Rim/patologia , Macrófagos/enzimologia , Metaloendopeptidases/análise , Adulto , Carcinoma de Células Renais/irrigação sanguínea , Carcinoma de Células Renais/patologia , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Receptores ErbB/análise , Proteínas Ligadas por GPI/análise , Humanos , Imuno-Histoquímica , Rim/irrigação sanguínea , Neoplasias Renais/irrigação sanguínea , Neoplasias Renais/patologia , Macrófagos/patologia , Pessoa de Meia-Idade , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologia , Análise Serial de Tecidos
16.
Clin Chim Acta ; 415: 306-16, 2013 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-23178445

RESUMO

This review covers carboxypeptidase M (CPM) research that appeared in the literature since 2009. The focus is on aspects that are new or interesting from a clinical perspective. Available research tools are discussed as well as their pitfalls and limitations. Evidence is provided to suggest the potential involvement of CPM in apoptosis, adipogenesis and cancer. This evidence derives from the expression pattern of CPM and its putative substrates in cells and tissues. In recent years CPM emerged as a potential cancer biomarker, in well differentiated liposarcoma where the CPM gene is co-amplified with the oncogene MDM2; and in lung adenocarcinoma where coexpression with EGFR correlates with poor prognosis. The available data call for extended investigation of the function of CPM in tumor cells, tumor-associated macrophages, stromal cells and tumor neovascularisation. Such experiments could be instrumental to validate CPM as a therapeutic target.


Assuntos
Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/enzimologia , Biomarcadores Tumorais/metabolismo , Lipossarcoma/irrigação sanguínea , Lipossarcoma/enzimologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/enzimologia , Metaloendopeptidases/metabolismo , Neoplasias de Tecidos Moles/irrigação sanguínea , Neoplasias de Tecidos Moles/enzimologia , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Adipogenia/genética , Apoptose/genética , Biomarcadores Tumorais/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Humanos , Lipossarcoma/diagnóstico , Lipossarcoma/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Metaloendopeptidases/genética , Neovascularização Patológica , Prognóstico , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Neoplasias de Tecidos Moles/diagnóstico , Neoplasias de Tecidos Moles/genética , Especificidade por Substrato
17.
Front Biosci (Landmark Ed) ; 16(7): 2427-50, 2011 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-21622187

RESUMO

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.


Assuntos
Carboxipeptidase B2/sangue , Fibrinólise/fisiologia , Coagulação Sanguínea/fisiologia , Carboxipeptidase B2/antagonistas & inibidores , Carboxipeptidase B2/química , Carboxipeptidase B2/genética , Desenho de Fármacos , Ativação Enzimática , Precursores Enzimáticos/sangue , Estabilidade Enzimática , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/farmacologia , Humanos , Modelos Biológicos , Trombose/sangue , Trombose/enzimologia
18.
Anal Biochem ; 403(1-2): 114-6, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20371219

RESUMO

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.


Assuntos
Carboxipeptidase B2/sangue , Bioquímica/métodos , Carboxipeptidase B2/metabolismo , Dipeptídeos/metabolismo , Humanos , Limite de Detecção , Modelos Lineares , Acidente Vascular Cerebral/tratamento farmacológico , Terapia Trombolítica , Ativador de Plasminogênio Tecidual/uso terapêutico
19.
Srp Arh Celok Lek ; 138 Suppl 1: 74-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20229688

RESUMO

Although the maintenance of precise balance between coagulation and fibrinolysis is of utmost importance for normal haemostasis, until recently these two systems were considered as completely separate mechanisms involved in the process of formation and dissolution of blood clot. Thrombin activatable fibrinolysis inhibitor (TAFI) is a recently described attenuator of the fibrinolytic rate and is considered to be the molecular link between coagulation and fibrinolysis. TAFI circulates in plasma as an inactive precursor and its conversion in active enzyme (TAFIa) occurs by the action of thrombin or plasmin, but most efficiently by thrombin in the presence of its cofactor thrombomodulin. Once generated, TAFI down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin; thereby preventing the upregulation of plasminogen binding and activation. Because TAFI is activated by thrombin on one side, and acts as the attenuator of fibrinolysis on another side, it enables fine synchronization between these two systems. The antifibrinolytic function of TAFI mostly depends on TAFI concentration, the rate of its activation and the half-life of TAFIa in plasma. Changes in thrombin generation can have a profound effect on the rate of TAFI activation, and consequently on the rate of fibrinolysis. Therefore, it has been hypothesized that increased thrombin generation seen in thrombophilia patients may enhance TAFI activation, leading to a hypofibrinolytic state, which may further contribute to the thrombotic tendency. However, the results of several studies, in which relation between TAFI level and the occurrence of thromboembolic complications in carriers of hereditary thrombophilia have been investigated, were not consistent.


Assuntos
Coagulação Sanguínea/fisiologia , Carboxipeptidase B2/fisiologia , Fibrinólise/fisiologia , Carboxipeptidase B2/sangue , Humanos , Fatores de Risco , Trombose/sangue
20.
Anal Biochem ; 396(1): 152-4, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19732738

RESUMO

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.


Assuntos
Carboxipeptidase B2/sangue , Ensaios Enzimáticos/métodos , Calibragem , Cromatografia Líquida de Alta Pressão , Humanos , Lisina Carboxipeptidase/sangue , Padrões de Referência , Especificidade por Substrato
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