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1.
Neth Heart J ; 27(10): 506-512, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31111455

RESUMO

INTRODUCTION: An increased body mass index (BMI) (>25 kg/m2) is associated with a wide range of electrocardiographic changes. However, the association between electrocardiographic changes and BMI in healthy young individuals with a normal BMI (18.5-25 kg/m2) is unknown. The aim of this study was to evaluate the association between BMI and electrocardiographic parameters. METHODS: Data from 1,290 volunteers aged 18 to 30 years collected at our centre were analysed. Only subjects considered healthy by a physician after review of collected data with a normal BMI and in sinus rhythm were included in the analysis. Subjects with a normal BMI (18.5-25 kg/m2) were divided into BMI quartiles analysis and a backward multivariate regression analysis with a normal BMI as a continuous variable was performed. RESULTS: Mean age was 22.7 ± 3.0 years, mean BMI was 22.0, and 73.4% were male. There were significant differences between the BMI quartiles in terms of maximum P-wave duration, P-wave balance, total P-wave area in lead V1, PR-interval duration, and heart axis. In the multivariate model maximum P-wave duration (standardised coefficient (SC) = +0.112, P < 0.001), P-wave balance in lead V1 (SC = +0.072, P < 0.001), heart axis (SC = -0.164, P < 0.001), and Sokolow-Lyon voltage (SC = -0.097, P < 0.001) were independently associated with BMI. CONCLUSION: Increased BMI was related with discrete electrocardiographic alterations including an increased P-wave duration, increased P-wave balance, a leftward shift of the heart axis, and decreased Sokolow-Lyon voltage on a standard twelve lead electrocardiogram in healthy young individuals with a normal BMI.

6.
Neth Heart J ; 20(3): 133-7, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22351557

RESUMO

To reduce long-term morbidity after revascularised acute myocardial infarction, different therapeutic strategies have been investigated. Cell therapy with mononuclear cells from bone marrow (BMMC) or peripheral blood (PBMC) has been proposed to attenuate the adverse processes of remodelling and subsequent heart failure. Previous trials have suggested that cell therapy may facilitate arrhythmogenesis. In the present substudy of the HEBE cell therapy trial, we investigated whether intracoronary cell therapy alters the prevalence of ventricular arrhythmias after 1 month or the rate of severe arrhythmogenic events (SAE) in the first year. In 164 patients of the trial we measured function and infarct size with cardiovascular magnetic resonance (CMR) imaging. Holter registration was performed after 1 month from which the number of triplets (3 successive PVCs) and ventricular tachycardias (VT, ≥4 successive PVCs) was assessed. Thirty-three patients (20%) showed triplets and/or VTs, with similar distribution amongst the groups (triplets: control n = 8 vs. BMMC n = 9, p = 1.00; vs. PBMC n = 10, p = 0.67. VT: control n = 9 vs. BMMC n = 9, p = 0.80; vs. PBMC n = 11, p = 0.69). SAE occurred in 2 patients in the PBMC group and 1 patient in the control group. In conclusion, intracoronary cell therapy is not associated with an increase in ventricular arrhythmias or SAE.

7.
Br J Clin Pharmacol ; 65(1): 22-9, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17610527

RESUMO

AIM: To study the pharmacokinetics (PK), safety and tolerability of single rising doses up to 80 mg of superoxide dismutase covalently linked to lecithin (PC-SOD) in healthy White volunteers. METHODS: This double-blind, placebo-controlled, four-period cross-over study was performed in eight healthy volunteers (four male/four female). Three doses of PC-SOD (20, 40 and 80 mg) and placebo were administered intravenously in randomized order. Serum and urinary PC-SOD concentrations were measured predose and up to 96 h after dosing. In addition to standard safety measurements, the urinary excretion of N-acetyl-beta-glucosaminidase, alpha-glutathione S-transferase (alpha-GST) and pi-GST was measured to evaluate renal function. The PK of PC-SOD was analysed using noncompartmental and compartmental methods. RESULTS: All treatments were well tolerated, and no obvious relationship between adverse events and treatment was observed. No effects of PC-SOD on renal function could be detected. Dose normalized C(max) and AUC were not different between the different dosages, indicating linearity of plasma concentrations with dose. Estimated PC-SOD clearance was 2.54 ml min(-1)[95% confidence interval (CI) 2.07, 2.83]. The terminal half-life was estimated to be 1.54 days (95% CI 0.93, 2.15). SOD activity was elevated above baseline for 19 +/- 6 h after the 80-mg dose. CONCLUSIONS: Single intravenous administrations of PC-SOD in doses up to 80 mg were well tolerated in healthy White male and female volunteers. With the doses used, SOD activity was linearly related to the dose; after the 80-mg dose it was present for an appreciable period. These findings suggest that it is worthwhile to investigate PC-SOD in clinical conditions characterized by a high radical overload.


Assuntos
Sequestradores de Radicais Livres/administração & dosagem , Sequestradores de Radicais Livres/farmacocinética , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/administração & dosagem , Superóxido Dismutase/farmacocinética , Adolescente , Adulto , Área Sob a Curva , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Método Duplo-Cego , Feminino , Sequestradores de Radicais Livres/farmacologia , Humanos , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Superóxido Dismutase/farmacologia
8.
Blood Coagul Fibrinolysis ; 12(7): 583-91, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11685048

RESUMO

During studies on warfarin, heparin and various anticoagulants with novel mechanisms of action, the activated partial thromboplastin time (aPTT) and the (apparent) international normalized ratio (INR) from a bedside monitor (Coagucheck Plus(R)) were compared with laboratory assay results. Data were compared using the Bland and Altman method of comparison where systematic differences result in significant slopes of the regression line. During heparin treatment, the bedside monitor largely underestimated the aPTT (slope = -0.80). During treatment with the direct thrombin inhibitor napsagatran (slope = 0.99), the pentasaccharides Org31540/SR90107A (slope = 0.77) and SanOrg34006 (slope = 0.35), and warfarin (slope = 0.60), the bedside monitor underestimated the aPTT at lower aPTT levels, while at higher aPTT levels it overestimated the laboratory values. The bedside monitor slightly overestimated the INR during treatment with warfarin (slope = 0.33). Apparent INR was largely overestimated during treatment with Org31540/SR90107A (slope = 1.38), SanOrg34006 (slope = 0.97), Napsagatran (slope = 1.23), and recombinant tissue factor pathway inhibitor (slope = 1.48, P < 0.001 for all regression lines). These results indicate that a substantial disagreement in aPTT or (apparent) INR exists between the bedside monitor and laboratory assay during treatment with the studied 'classic' and novel anticoagulants. The amount of disagreement depended on the anticoagulant given.


Assuntos
Anticoagulantes/uso terapêutico , Laboratórios , Tempo de Tromboplastina Parcial , Sistemas Automatizados de Assistência Junto ao Leito , Antitrombinas/uso terapêutico , Monitoramento de Medicamentos/métodos , Reações Falso-Negativas , Reações Falso-Positivas , Fibrinolíticos/uso terapêutico , Heparina/uso terapêutico , Humanos , Naftalenos/uso terapêutico , Oligossacarídeos/uso terapêutico , Piperidinas/uso terapêutico , Reprodutibilidade dos Testes , Varfarina/uso terapêutico
9.
Am J Physiol Regul Integr Comp Physiol ; 281(4): R1059-70, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11557611

RESUMO

We used cardiopulmonary bypass (CPB) as a model of activation of the contact system and investigated the involvement of the plasma and tissue kallikrein-kinin systems (KKS) in this process. Circulating levels of bradykinin and kallidin and their metabolites, plasma and tissue kallikrein, low and high molecular weight kininogen, and kallistatin were measured before, during, and 1, 4, and 10 h after CPB in subjects undergoing cardiac surgery. Bradykinin peptide levels increased 10- to 20-fold during the first 10 min, returned toward basal levels by 70 min of CPB, and remained 1.2- to 2.5-fold elevated after CPB. Kallidin peptide levels showed little change during CPB, but they were elevated 1.7- to 5.2-fold after CPB. There were reductions of 80 and 60% in plasma and tissue kallikrein levels, respectively, during the first minute of CPB. Kininogen and kallistatin levels were unchanged. Angiotensin-converting enzyme inhibition did not amplify the increase in bradykinin levels during CPB. Aprotinin administration prevented activation of the KKS. The changes in circulating kinin and kallikrein levels indicate activation of both the plasma and tissue KKS during activation of the contact system by CPB.


Assuntos
Ponte Cardiopulmonar , Cardiopatias/sangue , Cininas/sangue , Calicreína Plasmática/metabolismo , Calicreínas Teciduais/sangue , Angiotensina I/sangue , Angiotensina II/sangue , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Artérias , Proteínas Sanguíneas/metabolismo , Bradicinina/sangue , Proteínas de Transporte/sangue , Ativação Enzimática/efeitos dos fármacos , Cardiopatias/cirurgia , Humanos , Calidina/sangue , Cininogênios/sangue , Serpinas/sangue , Veias
10.
Thromb Res ; 103(1): 9-16, 2001 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-11434941

RESUMO

Dietary effects on liver blood flow may have biased the previously observed effects of hypertriglyceridemia on systemic tissue-type plasminogen activator (t-PA) concentrations. Therefore, in this study the effects of hypertriglyceridemia on plasma t-PA were determined by inducing hypertriglyceridemia with an intravenous fat emulsion (Intralipid) infusion. In a randomised crossover fashion, eight healthy male volunteers received Intralipid 10% (1.5 ml/min) or 0.9% saline for 2 h and 45 min. After 2 h of infusion, t-PA antigen, t-PA activity, t-PA/plasminogen activator inhibitor (PAI-1) complex, and PAI-1 activity were determined. Concomitantly, the effects of Intralipid t-PA clearance were determined from steady-state t-PA antigen concentrations of a 45-min recombinant tissue-type plasminogen activator (rt-PA) infusion (31.25 microg/min). Liver blood flow was assessed from steady-state concentrations of a continuous sorbitol infusion. Differences between treatments were calculated using the prevalue as the covariate. No significant differences were observed in mean+/-S.D. endogenous concentrations of t-PA antigen, 4.5+/-0.9/4.1+/-0.9 ng/ml (Intralipid vs. saline infusion; difference of 0.3 ng/ml, 95% confidence interval, CI: -0.2, 0.8); t-PA activity, 0.69+/-0.21/0.68+/-0.21 U/ml (difference of 0.04 U/ml, CI: -0.17, 0.25); t-PA/PAI-1 complex, 2.0+/-1.3/1.6+/-1.0 ng/ml (difference of 0.1 ng/ml, CI: -0.8, 0.6); and PAI-1 activity, 7.3+/-5.1/7.1+/-5.1 U/ml (difference of 0.26 U/ml, CI: -3.7, 4.3). Mean t-PA clearance and liver blood flow were unaffected by the Intralipid infusion. These results indicate that acute hypertriglyceridemia does not alter plasma fibrinolytic parameters in healthy male volunteers.


Assuntos
Hipertrigliceridemia/sangue , Ativador de Plasminogênio Tecidual/sangue , Doença Aguda , Adulto , Estudos Cross-Over , Emulsões Gordurosas Intravenosas/administração & dosagem , Emulsões Gordurosas Intravenosas/farmacologia , Humanos , Circulação Hepática , Masculino , Taxa de Depuração Metabólica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinética , Fluxo Sanguíneo Regional , Ativador de Plasminogênio Tecidual/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/farmacocinética
11.
Pharm Res ; 18(5): 593-9, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11465413

RESUMO

PURPOSE: The aim of the study was to investigate the cutaneous bioequivalence of a lipophilic model drug (lidocaine) applied in a novel topical microemulsion vehicle, compared to a conventional oil-in-water (O/W) emulsion, assessed by a pharmacokinetics microdialysis model and a pharmacodynamic method. METHODS: Dermal delivery of lidocaine was estimated by microdialysis in 8 volunteers. Absorption coefficients and lag times were determined by pharmacokinetic modelling of the microdialysis data. Subsequently, the anaesthetic effect of the treatments was assessed by mechanical stimuli using von Frey hairs in 12 volunteers. RESULTS: The microemulsion formulation increased the cutaneous absorption coefficient of lidocaine 2.9 times (95% confidence interval: 1.9/4.6) compared with the O/W emulsion-based cream. Also, lag time decreased from 110 +/- 43 min to 87 +/- 32 min (P = 0.02). The compartmental pharmacokinetic model provided an excellent fit of the concentration-time curves with reliable estimation of absorption coefficient and lag time. A significant anaesthetic effect was found for both active treatments compared to placebo (P < 0.02), but the effect did not diverge significantly between the two formulations. CONCLUSIONS: The microemulsion vehicle can be applied to increase dermal drug delivery of lipophilic drugs in humans. The microdialysis technique combined with an appropriate pharmacokinetic model provides a high sensitivity in bioequivalence studies of topically applied substances.


Assuntos
Anestésicos Locais/administração & dosagem , Anestésicos Locais/farmacocinética , Lidocaína/administração & dosagem , Lidocaína/farmacocinética , Veículos Farmacêuticos , Absorção Cutânea/fisiologia , Administração Tópica , Adulto , Algoritmos , Anestésicos Locais/química , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Emulsões , Humanos , Lidocaína/química , Masculino , Microdiálise , Medição da Dor , Equivalência Terapêutica
12.
J Cardiovasc Pharmacol ; 36(6): 699-703, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11117368

RESUMO

The receptor involved in the serotonin (5-hydroxytryptamine [5-HT])-induced vasodilatation in the human forearm has not yet been identified. Experimental data point to the 5-HT2B receptor located on the endothelium. RS-127445 (2-amino-4-(4-fluoronaphthyl-1-yl)-6-isopropylpyrimidine) is a novel potent and selective 5-HT2B receptor antagonist. The effect of oral RS-127445 (500 mg) on 5-HT-induced vasodilatation was studied in a double-blind, randomized, placebo-controlled, crossover study in six healthy volunteers. On each study day 5-HT (0.5 ng/kg/min) was infused into the brachial artery for 8 min, before drug administration and at intervals of 20, 65, 110, 230, and 470 min after oral ingestion. At each infusion, plasma samples for study drug assay were taken and forearm blood flow was assessed using venous occlusion plethysmography. Although (log) drug concentrations exceeded pKi, there was no correlation between RS-127445 concentrations and 5-HT-induced vasodilatation. 5-HT-induced vasodilatation did not differ between treatments and time points. It appears that there is no functional involvement of 5-HT2B receptors in 5-HT-mediated vasodilatation in the human forearm.


Assuntos
Antebraço/irrigação sanguínea , Serotonina/farmacologia , Vasodilatação/efeitos dos fármacos , Adulto , Estudos Cross-Over , Método Duplo-Cego , Humanos , Masculino , Pirimidinas/farmacologia , Receptor 5-HT2B de Serotonina , Receptores de Serotonina/fisiologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia
13.
J Immunoassay ; 21(4): 411-26, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11071257

RESUMO

Human tissue prokallikrein is the enzymatically inactive zymogen of a serine proteinase involved in the liberation of vasoactive kinin peptides, and it is supposed that an impaired prokallikrein-to-kallikrein conversion is closely related to certain hypertensive and inflammatory disorders. Progress in understanding the biological role of the proenzyme has been limited by the absence of an accurate assay for the kallikrein precursor. We describe a sandwich enzyme-linked immunosorbent assay to measure human tissue prokallikrein using monospecific anti-peptide antibodies raised against propeptide derivatives. This method could detect a minimum concentration of 60 pg/ml prokallikrein and displayed no cross-reactivity or interference with mature tissue kallikrein. The intra- and inter-assay precision varied from 8-15%, respectively, indicating a reasonable reproducibility of the method. The level of prokallikrein was defined in different human urine samples, and the corresponding dilution curves showed good linearity. The mean recovery of added zymogen was 104%. Prokallikrein immunoassay is the first reported tool for the direct and sensitive quantification of the precursor of tissue kallikrein and should facilitate the precise determination of prokallikrein levels in a variety of biological specimen.


Assuntos
Precursores Enzimáticos/urina , Ensaio de Imunoadsorção Enzimática/métodos , Calicreínas/urina , Adulto , Reações Cruzadas , Precursores Enzimáticos/imunologia , Humanos , Calicreínas/imunologia , Fragmentos de Peptídeos/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
14.
Biol Chem ; 381(7): 629-32, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10987372

RESUMO

The kallikrein-kinin system is a mediator of inflammation in humans. In order to elucidate the range of expression of human tissue kallikrein and its substrates, high and low molecular weight kininogen, in inflammatory cells in vitro, we examined their biosynthesis in the HL-60 cell line by RT-PCR and Southern blot analyses. Prominent expression of tissue kallikrein mRNA occurred in untreated promyelocytic cultures as well as in HL-60 cells that were induced to differentiate toward neutrophilic, monocytic, and macrophagic cells. Under the same inducing conditions, kininogen biosynthesis was undetectable at each differentiation state of HL-60 cultures. These results indicate that the myelomonocytic lineage of human leukocytes is a source of tissue kallikrein, which may be secreted as part of the inflammatory process.


Assuntos
Calicreínas Teciduais/genética , Sequência de Bases , Southern Blotting , Diferenciação Celular , Primers do DNA , Células HL-60 , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Clin Pharmacol Ther ; 67(5): 504-11, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10824629

RESUMO

OBJECTIVES: Recombinant tissue factor pathway inhibitor (rTFPI) has been shown to be an effective treatment in animal models of sepsis and is under investigation for human use. Reduced liver blood flow during septic shock may substantially alter the pharmacokinetics of rTFPI because clearance of rTFPI approaches liver blood flow. The aim of this study was to examine the effect of exercise-induced reduction in liver blood flow on the pharmacokinetics and pharmacodynamics of rTFPI. METHODS: This was a two-way, open-label, randomized crossover study in eight healthy male volunteers. The subjects in both treatment groups received a continuous intravenous infusion of rTFPI (0.2 mg/kg/h) concurrently with intravenous sorbitol (50 mg/min) for 4 hours. Sorbitol was used as a biomarker for liver blood flow. The subjects were randomized to remain supine or to exercise on a bicycle ergometer for 30 minutes starting at the beginning of the third hour of the infusion. RESULTS: Exercise reduced liver blood flow (mean +/- SEM) from 1.44 +/- 0.06 L/min to 0.40 +/- 0.03 L/min. The average clearance of rTFPI decreased from 0.73 +/- 0.04 L/min in the supine position to 0.25 +/- 0.02 L/min during exercise. This decrease in rTFPI clearance resulted in an 80% (95% confidence interval [CI], 60% to 102%) increase in plasma rTFPI levels during exercise. The average maximal prothrombin time and activated partial thromboplastin time values during exercise were 1.4 (95% CI, 0.4 to 2.5) and 4.4 (95% CI, 2.7 to 6.1) seconds higher compared with the supine steady-state level. CONCLUSIONS: Reduction in liver blood flow by exercise markedly increased rTFPI concentrations and induced a slight but variable prothrombin time and activated partial thromboplastin time increase at the rTFPI dose studied.


Assuntos
Anticoagulantes/farmacocinética , Inibidores do Fator Xa , Lipoproteínas/farmacocinética , Fígado/irrigação sanguínea , Fígado/metabolismo , Adulto , Anticoagulantes/sangue , Velocidade do Fluxo Sanguíneo/fisiologia , Estudos Cross-Over , Exercício Físico/fisiologia , Humanos , Infusões Intravenosas , Lipoproteínas/sangue , Masculino , Tempo de Tromboplastina Parcial , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinética , Valores de Referência , Choque Séptico/metabolismo , Choque Séptico/fisiopatologia , Sorbitol/sangue , Decúbito Dorsal
16.
FEBS Lett ; 467(2-3): 165-8, 2000 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-10675531

RESUMO

Human tissue prokallikrein, a zymogen of the kallikrein-kinin system, circulates in plasma bound to neutrophils. Because plasma kininogens contribute to the assembly of kinin-generating components on blood cells, these proteins were assessed for their ability to complex the kallikrein precursor. Using ligand blot and direct binding assays, biotinylated prokallikrein was found to bind only to high-molecular-weight kininogen and not to the low-molecular-weight form. The interaction was specific, reversible, and saturable yielding an estimated dissociation constant K(D)=690 nM and a 1:1 stoichiometry. Specific kininogen binding of tissue prokallikrein also occurred at physiological plasma protein concentrations. These results provide the first evidence for a novel function of high-molecular-weight kininogen as a binding protein for tissue prokallikrein that could serve to localize the kallikrein precursor on the neutrophil surface.


Assuntos
Proteínas de Transporte/química , Precursores Enzimáticos/química , Calicreínas/química , Cininogênio de Alto Peso Molecular/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Precursores Enzimáticos/metabolismo , Humanos , Calicreínas/metabolismo , Cininogênio de Alto Peso Molecular/metabolismo , Neutrófilos/metabolismo
17.
Br J Clin Pharmacol ; 50(6): 621-3, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11136302

RESUMO

AIMS: To determine the influence of the hand circulation on the determination of venous distensibility with venous occlusion plethysmography. METHODS: In a randomised study, duplicate measurements of forearm venous distensibility, with and without a wrist cuff, were made over occlusion periods of 3 and 12 min in eight volunteers. Treatments were compared with paired Student's t-tests and differences are presented as 95% confidence intervals (CI). Intra-subject variability was assessed with analysis of variance. RESULTS: Non-significant differences in increases in forearm volume between the occlusions with and without wrist cuff were found for the 3 min occlusion (CI: -0.4, + 0.2%) and the 12 min occlusion period (CI: -0.7, + 0.2%). However, the coefficient of variation was lower with the use of a wrist cuff; after 3 min occlusion (12% vs 19%) and after 12 min of occlusion (14% vs 24%). Forearm volume after 12 min of venous occlusion was 0.5% (CI: + 0.4, + 0.7) higher than after 3 min. CONCLUSIONS: Although venous distensibility was equal when assessed with and without wrist cuff, exclusion of the hand circulation reduces intraindividual variability. Equilibrium in forearm volume is not reached after 3 min period of venous occlusion, as often assumed. The magnitude of the additional increase after prolonged occlusion stresses the need for well-controlled studies.


Assuntos
Antebraço/irrigação sanguínea , Mãos/irrigação sanguínea , Capacitância Vascular/fisiologia , Veias/fisiologia , Adulto , Circulação Sanguínea , Antebraço/fisiologia , Humanos , Masculino , Pletismografia
18.
Immunopharmacology ; 45(1-3): 95-101, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10614996

RESUMO

The recent discovery of tissue prokallikrein (TproK) on the cell surface of human neutrophils prompted this study to test the sensitivity of the kallikrein precursor to proteolysis by neutrophil-derived elastase and cathepsin G. Purified recombinant human TproK was readily degraded by cathepsin G via distinct cleavage sites into two major fragments of 5 and 8 kDa which are devoid of enzymatic activity. Leukocyte elastase caused a rapid conversion of TproK into [des-Ile1]-TK, a differentially processed tissue kallikrein (TK) isoform with truncated N-terminus that appeared to be resistant to further cleavage by elastase. Kinetic data demonstrated the cleavage of amidolytic kallikrein substrates and the release of kinin from HMW kininogen, but the single amino acid deletion results in a severe reduction of catalytic activity for [des-Ile1]-TK compared to the mature enzyme. These in vitro observations support the possibility that the inter-relationships among TproK and neutrophil proteinases may provide a sensitive regulatory system to balance the kallikrein-kinin cascade during inflammatory events.


Assuntos
Precursores Enzimáticos/metabolismo , Calicreínas/metabolismo , Elastase de Leucócito/metabolismo , Neutrófilos/enzimologia , Catepsina G , Catepsinas/metabolismo , Humanos , Hidrólise , Processamento de Proteína Pós-Traducional , Serina Endopeptidases
19.
Biol Chem ; 380(11): 1321-8, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10614825

RESUMO

Putative binding sites for prokallikrein, the endogenous zymogen of the vasoactive and pro-inflammatory tissue kallikrein-kinin system, were recently demonstrated on human neutrophils. However, the occurrence and distribution of neutrophil-bound prokallikrein itself have so far not been examined. In this study, a specific anti-peptide antibody directed against the propart of the zymogen was used to localize the kallikrein precursor by confocal laser-scanning microscopy on unstimulated human blood neutrophils. Our results describe, for the first time, the presence of tissue prokallikrein on the membrane of circulating neutrophils. Immunoreactive prokallikrein was associated into punctate clusters occupying the external surface of the neutrophil membrane and, after addition of exogenous zymogen, immunolabeling was enhanced four-fold. In contrast, only moderate immunoreactivity to prokallikrein was observed intracellularly. These results suggest that resting neutrophils provide a circulating platform for tissue prokallikrein whose surface density may be upregulated as part of the inflammatory process.


Assuntos
Membrana Celular/metabolismo , Precursores Enzimáticos/imunologia , Precursores Enzimáticos/metabolismo , Calicreínas/imunologia , Calicreínas/metabolismo , Neutrófilos/metabolismo , Membrana Celular/imunologia , Precursores Enzimáticos/análise , Humanos , Calicreínas/análise , Masculino , Neutrófilos/imunologia , Coloração e Rotulagem/métodos
20.
Br J Haematol ; 105(3): 599-612, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10354119

RESUMO

The vasoactive peptides bradykinin and kallidin (lysyl-bradykinin) have been implicated in diapedesis, a cellular process by which neutrophils migrate through endothelial cell gap junctions. The kinin peptides are released from their precursor moiety, kininogen, by the specific action of endoproteinases, the kallikreins. Kininogens have been demonstrated on the surface of neutrophils, and the presence of a competent processing enzyme such as tissue prokallikrein in neutrophils has been postulated, but firm evidence for this is still lacking. We have raised antibodies to a synthetic peptide that is a sequence copy of the activation segment of human TK and demonstrated that the anti-peptide antibodies specifically recognized the zymogen but not the active form of kallikrein. Using these anti-peptide antibodies, we showed by Western blotting, immunocytochemistry and electron microscopy that the tissue prokallikrein antigen was localized in neutrophils and their precursor cells, the myelocytes. We further demonstrated by in situ hybridization the presence of tissue kallikrein mRNA in the mature neutrophils and myelocytes. Our findings lend credence to the hypothesis that upon release and activation, neutrophil-borne TK acts on cell-associated kininogens to trigger the release of kinins, which may open endothelial gates for neutrophil diapedesis.


Assuntos
Células da Medula Óssea/metabolismo , Precursores Enzimáticos/metabolismo , Calicreínas/metabolismo , Neutrófilos/metabolismo , Sequência de Bases , Western Blotting , Células da Medula Óssea/ultraestrutura , Humanos , Hibridização In Situ , Microscopia , Microscopia Eletrônica , Dados de Sequência Molecular , Neutrófilos/ultraestrutura , RNA Mensageiro/metabolismo
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