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BACKGROUND AND OBJECTIVES: Repeated blood donation produces iron deficiency. Changes in dietary iron intake do not prevent donation-induced iron deficiency. Prolonging the interdonation interval or using oral iron supplements can mitigate donation-induced iron deficiency. The most effective operational methods for reducing iron deficiency in donors are unknown. MATERIALS AND METHODS: 'Strategies To Reduce Iron Deficiency' (STRIDE) was a two-year, randomized, placebo-controlled study in blood donors. 692 donors were randomized into one of two educational groups or one of three interventional groups. Donors randomized to educational groups either received letters thanking them for donating, or, suggesting iron supplements or delayed donation if they had low ferritin. Donors randomized to interventional groups either received placebo, 19-mg or 38-mg iron pills. RESULTS: Iron deficient erythropoiesis was present in 52·7% of males and 74·6% of females at enrolment. Adverse events within 60 days of enrolment were primarily mild gastrointestinal symptoms (64%). The incidence of de-enrolment within 60 days was more common in the interventional groups than in the educational groups (P = 0·002), but not more common in those receiving iron than placebo (P = 0·68). CONCLUSION: The prevalence of iron deficient erythropoiesis in donors enrolled in the STRIDE study is comparable to previously described cohorts of regular blood donors. De-enrolment within 60 days was higher for donors receiving tablets, although no more common in donors receiving iron than placebo.
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Anemia Ferropriva/prevenção & controle , Doadores de Sangue , Deficiências de Ferro , Ferro da Dieta/uso terapêutico , Adulto , Suplementos Nutricionais , Método Duplo-Cego , Eritropoese , Feminino , Humanos , Ferro/sangue , Ferro da Dieta/administração & dosagem , Ferro da Dieta/efeitos adversos , MasculinoRESUMO
Introduction: The present systematic review and meta-analysis explores the impacts of cognitive processing therapy (CPT), eye movement desensitization and reprocessing (EMDR), and prolonged exposure (PE) therapy on neural activity underlying the phenomenon of post-traumatic growth for adult trauma survivors. Methods: We utilized the following databases to conduct our systematic search: Boston College Libraries, PubMed, MEDLINE, and PsycINFO. Our initial search yielded 834 studies for initial screening. We implemented seven eligibility criteria to vet articles for full-text review. Twenty-nine studies remained for full-text review after our systematic review process was completed. Studies were subjected to several levels of analysis. First, pre-and post- test post-traumatic growth inventory (PTGI) scores were collected from all studies and analyzed through a forest plot using Hedges' g. Next, Montreal Neurological Institute (MNI) coordinates and t-scores were collected and analyzed using an Activation Likelihood Estimation (ALE) to measure brain function. T-scores and Hedges' g values were then analyzed using Pearson correlations to determine if there were any relationships between brain function and post-traumatic growth for each modality. Lastly, all studies were subjected to a bubble plot and Egger's test to assess risk of publication bias across the review sample. Results: Forest plot results indicated that all three interventions had a robust effect on PTGI scores. ALE meta-analysis results indicated that EMDR exhibited the largest effect on brain function, with the R thalamus (t = 4.23, p < 0.001) showing robust activation, followed closely by the R precuneus (t = 4.19, p < 0.001). Pearson correlation results showed that EMDR demonstrated the strongest correlation between increased brain function and PTGI scores (r = 0.910, p < 0.001). Qualitative review of the bubble plot indicated no obvious traces of publication bias, which was corroborated by the results of the Egger's test (p = 0.127). Discussion: Our systematic review and meta-analysis showed that CPT, EMDR, and PE each exhibited a robust effect on PTG impacts across the course of treatment. However, when looking closer at comparative analyses of neural activity (ALE) and PTGI scores (Pearson correlation), EMDR exhibited a more robust effect on PTG impacts and brain function than CPT and PE.
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BACKGROUND AND OBJECTIVE: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are endothelial-associated anticoagulant proteins thought to control hemostasis in specific vascular beds. Here, we have examined the consequences of TFPI deficiency in the presence of a compounding procoagulant state caused by reduced TM function. METHODS AND RESULTS: TFPI(+/-)/TM(pro/pro) mice are born at less than expected frequency in either TFPI(+/-)/TM(pro/+) or TM(pro/pro) mothers but are born at near the expected frequency in TM(pro/+) mothers. Adult TFPI(+/-)/TM(pro/pro) mice have elevated thrombin-antithrombin complex and increased thrombus volume in an electrical injury model of venous thrombosis. In striking contrast to mice with single deficiency of TFPI or TM, TFPI(+/-)/TM(pro/pro) mice exhibit augmented fibrin deposition not only in the liver, but also in the cerebral microvasculature. CONCLUSIONS: TFPI(+/-)/TM(pro/pro) mice exhibit partial intrauterine lethality when carried by mothers with an underlying prothrombotic state, providing the first experimental evidence in an animal model that TFPI-dependent control of hemostasis in the vascular bed of the placenta fulfills a critical role for successful pregnancy outcome. In addition to the placenta, partial TFPI deficiency interacts with decreased TM function in an organ selective manner to produce fibrin deposition in other specific vascular beds, the liver and brain.
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Fibrina/metabolismo , Lipoproteínas/deficiência , Trombomodulina/deficiência , Trombofilia/etiologia , Animais , Circulação Cerebrovascular , Feminino , Genótipo , Lipoproteínas/genética , Fígado/irrigação sanguínea , Camundongos , Microcirculação , Especificidade de Órgãos , Placenta/irrigação sanguínea , Gravidez , Resultado da Gravidez , Trombomodulina/genéticaRESUMO
Airway mesenchymal cells, such as myofibroblasts and airway smooth muscle cells, contribute to inflammation, airway remodelling and hyperresponsiveness in asthma by excessive proliferation and inflammatory mediator production. Using endobronchial biopsies obtained from both nonasthmatic and asthmatic subjects, in situ proliferation was assessed by immunostaining for cyclin D1. The number of immunoreactive cells increased with asthma severity and was restricted to the epithelium and subepithelial connective tissue. Despite increases in smooth muscle area, cyclin D1 was not detected in cells in intact muscle bundles. Biopsy-derived cell cultures were characterised as predominantly myofibroblasts, and were assessed to determine whether proliferation and cytokine production varied with asthma status. Cell enumeration showed that basal proliferation was similar in cells from nonasthmatics and asthmatics, and mitogenic responses to fibroblast growth factor-2, thrombin or serum were either reduced or unchanged in cells from asthmatics. Interleukin (IL)-1-dependent granulocyte-macrophage colony-stimulating factor and IL-8 release was increased in cell supernatants from asthmatics. Thus, increased rates of cellular proliferation identified in situ in the asthmatic airway occurred outside the expanded smooth muscle compartment. Although reduced proliferative responses were observed in cultured myofibroblasts from asthmatics, the increased cytokine production by these cells suggests that this contributes to and may perpetuate ongoing inflammation in asthma.
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Albuterol/análogos & derivados , Androstadienos/farmacologia , Asma/metabolismo , Fibroblastos/metabolismo , Músculo Liso/metabolismo , Músculos/metabolismo , Traqueia/metabolismo , Adulto , Albuterol/farmacologia , Broncodilatadores/farmacologia , Proliferação de Células , Ciclina D1/metabolismo , Feminino , Fluticasona , Humanos , Masculino , Pessoa de Meia-Idade , Xinafoato de SalmeterolRESUMO
Essentials Heparin-protamine balance (HPB) modulates bleeding after neonatal cardiopulmonary bypass (CPB). HPB was examined in 44 neonates undergoing CPB. Post-operative bleeding occurred in 36% and heparin rebound in 73%. Thrombin-initiated fibrin clot kinetic assay and partial thromboplastin time best assessed HPB. SUMMARY: Background Neonates undergoing cardiopulmonary bypass (CPB) are at risk of excessive bleeding. Blood is anticoagulated with heparin during CPB. Heparin activity is reversed with protamine at the end of CPB. Paradoxically, protamine also inhibits blood coagulation when it is dosed in excess of heparin. Objectives To evaluate heparin-protamine balance in neonates undergoing CPB by using research and clinical assays, and to determine its association with postoperative bleeding. Patients/Methods Neonates undergoing CPB in the first 30 days of life were studied. Blood samples were obtained during and after surgery. Heparin-protamine balance was assessed with calibrated automated thrombography, thrombin-initiated fibrin clot kinetic assay (TFCK), activated partial thromboplastin time (APTT), anti-FXa activity, and thromboelastometry. Excessive postoperative bleeding was determined by measurement of chest tube output or the development of cardiac tamponade. Results and Conclusions Of 44 neonates enrolled, 16 (36%) had excessive postoperative bleeding. The TFCK value was increased. By heparin in neonatal blood samples, but was only minimally altered by excess protamine. Therefore, it reliably measured heparin in samples containing a wide range of heparin and protamine concentrations. The APTT most closely correlated with TFCK results, whereas anti-FXa and thromboelastometry assays were less correlative. The TFCK and APTT assay also consistently detected postoperative heparin rebound, providing an important continued role for these long-established coagulation tests in the management of postoperative bleeding in neonates requiring cardiac surgical repair. None of the coagulation tests predicted the neonates who experienced postoperative bleeding, reflecting the multifactorial causes of bleeding in this population.
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Anticoagulantes/administração & dosagem , Coagulação Sanguínea/efeitos dos fármacos , Ponte Cardiopulmonar/efeitos adversos , Antagonistas de Heparina/administração & dosagem , Heparina/administração & dosagem , Hemorragia Pós-Operatória/etiologia , Protaminas/administração & dosagem , Anticoagulantes/efeitos adversos , Anticoagulantes/sangue , Testes de Coagulação Sanguínea , Monitoramento de Medicamentos/métodos , Feminino , Heparina/efeitos adversos , Heparina/sangue , Antagonistas de Heparina/efeitos adversos , Antagonistas de Heparina/sangue , Humanos , Recém-Nascido , Masculino , Hemorragia Pós-Operatória/sangue , Hemorragia Pós-Operatória/diagnóstico , Valor Preditivo dos Testes , Estudos Prospectivos , Protaminas/efeitos adversos , Protaminas/sangue , Fatores de Risco , Resultado do TratamentoRESUMO
Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that directly inhibits coagulation Factor Xa and also inhibits tissue factor-initiated coagulation. Normal human plasma TFPI exists both as the full-length molecule and as variably carboxy-terminal truncated forms. We reported recently that the low density lipoprotein receptor-related protein mediates the cellular degradation of TFPI after TFPI binding to the hepatoma cell surface. To examine whether the carboxy terminus of TFPI was required for interacting with hepatoma cells, a mutant of TFPI lacking the third Kunitz-type domain and basic carboxy terminus was generated. We found that this mutant, TFPI-160, did not compete with full-length 125I-TFPI-160 for binding to hepatoma cells. We were also unable to demonstrate specific binding of 125I-TFPI-160 to hepatoma cells at 4 degrees C. At 37 degrees C, significantly less 125I-TFPI-160 was internalized and degraded via low density lipoprotein receptor-related protein than full-length 125I-TFPI. Full-length 125I-TFPI binding to hepatoma cells could be inhibited > 90% by heparin and other highly charged molecules. Since TFPI, but not TFPI-160, was capable of effectively binding to cultured hepatoma cells, the fates of TFPI and TFPI-160 in vivo were examined. Both 125I-TFPI and 125I-TFPI-160 disappeared rapidly from the circulation after their intravenous administration into rats. The initial plasma half-life of 125I-TFPI was approximately 30 s whereas the half-life of 125I-TFPI-160 was approximately 4 min. 125I-TFPI was cleared predominantly by the liver. In contrast, 125I-TFPI-160 accumulated in the outer cortex of the kidney. Using microscopic autoradiography, we demonstrate that 125I-TFPI clearance is largely hepatocellular, whereas 125I-TFPI-160 accumulates mainly in the cells of the kidney proximal tubules. Together our findings demonstrate that the carboxy-terminal region(s) distal to amino acid 160 of TFPI mediates TFPI binding to hepatoma cells both in vitro and in vivo.
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Carcinoma Hepatocelular/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/metabolismo , Neoplasias Hepáticas/metabolismo , Inibidores de Serina Proteinase/metabolismo , Animais , Ligação Competitiva , Transporte Biológico , Humanos , Rim/metabolismo , Lipoproteínas/farmacocinética , Fígado/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Taxa de Depuração Metabólica , Poliaminas/farmacologia , Polieletrólitos , Polímeros/farmacologia , Ligação Proteica , Ratos , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.
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DNA/metabolismo , Sondas de Oligonucleotídeos , Polimorfismo de Fragmento de Restrição , Archaeoglobus fulgidus/genética , Bacteriófago M13/genética , DNA/isolamento & purificação , Endonucleases/genética , Exodesoxirribonuclease V , Exodesoxirribonucleases/genética , Leucócitos/metabolismo , Modelos Biológicos , Mutagênese Insercional , Pyrococcus furiosus/genética , Espectrometria de FluorescênciaRESUMO
BACKGROUND: The tissue factor (TF) factor (F) VIIa complex activates coagulation FIX and FX to initiate coagulation, and also cleaves protease activated receptors (PARs) to initiate inflammatory processes in vascular cells. Tissue factor pathway inhibitor (TFPI) is the only specific inhibitor of the TF-FVIIa complex, regulating both its procoagulant and pro-inflammatory properties. Upon heparin infusion during cardiopulmonary bypass (CPB), a heparin releasable pool of endothelial associated TFPI circulates in plasma. Following protamine neutralization of heparin, the plasma TFPI level decreases, but does not return completely to baseline, suggesting that during CPB a fraction of the plasma TFPI becomes heparin-independent. We have investigated the structural and functional properties of plasma TFPI during CPB to further characterize how TFPI is altered during this procedure. METHODS: We enrolled 17 patients undergoing first-time cardiac surgery involving CPB. Plasma samples were obtained at baseline, 5 min and 1 h after start of CPB (receiving heparin), 10 min after protamine administration (off CPB) and 24 h following surgery. Samples were analyzed for full-length and free (non-lipoprotein bound) TFPI antigen by enzyme-linked immunosorbent assay (ELISA) and for TFPI anticoagulant activity using an amidolytic assay. Western blot analysis was used to identify TFPI species of varying molecular weights in three additional patients. Dunnett's test for post hoc comparisons was used for statistical analysis. RESULTS: The ELISA and Western blot data indicated that an increase in full-length TFPI accounted for most of the heparin releasable TFPI. Following heparin neutralization with protamine, the full-length TFPI antigen returned to baseline levels while the free TFPI antigen and the total plasma TFPI activity remained elevated. This was associated with the appearance of a new 38 kDa form of plasma TFPI identified by Western blot analysis. The 38 kDa form of TFPI did not react with an antibody directed against the C-terminal region of TFPI indicating it has undergone proteolysis within this region. All TFPI measurements returned to baseline 24 h following CPB. CONCLUSIONS: During CPB the full-length form of TFPI is the predominant form in plasma because of its prompt release from the endothelial surface following heparin administration. Upon heparin neutralization with protamine, full-length TFPI redistributes back to the endothelial surface. However, a new 38 kDa TFPI fragment is generated during CPB and remains circulating in plasma, indicating that TFPI undergoes proteolytic degradation during CPB. This degradation may result in a decrease in endothelium-associated TFPI immediately post-CPB, and may contribute to the procoagulant and proinflammatory state that often complicates CPB.
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Ponte de Artéria Coronária , Lipoproteínas/fisiologia , Adulto , Sequência de Aminoácidos , Anticorpos/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Antagonistas de Heparina/farmacologia , Humanos , Imunoprecipitação , Lipoproteínas/sangue , Lipoproteínas/química , Lipoproteínas/imunologia , Dados de Sequência Molecular , Protaminas/administração & dosagemRESUMO
BACKGROUND: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)-anchored co-receptor. OBJECTIVES/METHODS: Cellular trafficking of TFPI within aerolysin-resistant ECV304 and EA.hy926 cells, which do not express GPI-anchored proteins on their surface, was compared with their wild-type counterparts. RESULTS AND CONCLUSIONS: Although aerolysin-resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild-type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin-resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin-resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI-anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co-receptor in the ER/Golgi. The co-receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild-type cells or to lysosomes of aerolysin-resistant cells. TFPI that escapes co-receptor binding is secreted through the same pathway in both wild-type and aerolysin-resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.
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Glicosilfosfatidilinositóis/metabolismo , Lipoproteínas/metabolismo , Toxinas Bacterianas/farmacologia , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Lipoproteínas/genética , Reação em Cadeia da Polimerase , Proteínas Citotóxicas Formadoras de Poros , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The in vivo catabolism of 125I-labeled alpha 1-antichymotrypsin was studied in our previously described mouse model. Native alpha 1-antichymotrypsin cleared with an apparent t1/2 of 85 min, but alpha 1-antichymotrypsin in complex with chymotrypsin or cathepsin G cleared with a t1/2 of 12 min. Clearance of the complex was blocked by a large molar excess of unlabeled complexes of proteinases with either alpha 1-antichymotrypsin or alpha 1-proteinase inhibitor. These studies indicate that the clearance of alpha 1-antichymotrypsin-proteinase complexes utilizes the same pathway as complexes with the homologous inhibitor alpha 1-proteinase inhibitor. Previous studies have demonstrated that this pathway is also responsible for the catabolism of two other serine proteinase inhibitors, antithrombin III and heparin cofactor II. This pathway is thus responsible for removing several proteinases involved in coagulation and inflammation from the circulation, thereby decreasing the likelihood of adventitious proteolysis.
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Antitrombina III/metabolismo , Proteínas Sanguíneas/metabolismo , Catepsinas/metabolismo , Glicoproteínas/metabolismo , Inibidores de Proteases/metabolismo , alfa 1-Antiquimotripsina/metabolismo , Antitrombina III/farmacocinética , Sítios de Ligação , Proteínas Sanguíneas/farmacocinética , Catepsina G , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/farmacocinética , Meia-Vida , Cofator II da Heparina , Humanos , Serina Endopeptidases , alfa 1-Antiquimotripsina/farmacocinética , alfa 1-AntitripsinaRESUMO
Tissue factor pathway inhibitor (TFPI) dampens the initiation of blood coagulation by inhibiting two potent procoagulant complexes, tissue factor-factor VIIa (TF-FVIIa) and early forms of prothrombinase. TFPI isoforms, TFPIα and TFPIß, result from alternative splicing of mRNA, producing distinct C-terminal ends of the two proteins. Both isoforms inhibit TF-FVIIa, but only TFPIα can inhibit early forms of prothrombinase by binding of its positively charged C-terminus with high affinity to the acidic B-domain exosite of FVa, which is generated upon activation by FXa. TFPIα and TFPIß are produced in cultured human endothelial cells, while platelets contain only TFPIα. Knowledge of the anticoagulant mechanisms and tissue expression patterns of TFPIα and TFPIß have improved our understanding of the phenotypes observed in different mouse models of TFPI deficiency, the east Texas bleeding disorder, and the development of pharmaceutical agents that block TFPI function to treat hemophilia.
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Coagulação Sanguínea , Lipoproteínas/metabolismo , Animais , Transtornos Herdados da Coagulação Sanguínea/sangue , Transtornos Herdados da Coagulação Sanguínea/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Hemofilia A/sangue , Hemofilia A/tratamento farmacológico , Hemofilia A/genética , Hemorragia/genética , Hemorragia/prevenção & controle , Hemostasia/efeitos dos fármacos , Hemostáticos/uso terapêutico , Humanos , Lipoproteínas/antagonistas & inibidores , Lipoproteínas/química , Lipoproteínas/deficiência , Lipoproteínas/genética , Fenótipo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: A delay in gastric emptying rate has been reported in peritoneal dialysis patients, often normalizing after evacuation of the dialysate. To evaluate the effect of the intraperitoneal volume, we compared this finding with a cirrhotic model in which gastric emptying was studied before and after a large-volume paracentesis. METHODS AND DESIGN: We used the 13C-octanoic acid breath test to measure gastric half-emptying time (T1/2) for solids in patients with alcoholic cirrhosis, non-diabetic peritoneal dialysis patients, and a control population (asymptomatic volunteers). Cirrhotic patients underwent the test on two consecutive mornings before and after an evacuating paracentesis. Peritoneal dialysis patients were studied twice on consecutive days: once with the dialysate present intra-abdominally ("full"), and once with an emptied abdomen ("empty"). Biochemical analysis was carried out on blood samples before the first test. All cirrhotics underwent a 13C-aminopyrine breath test to assess residual liver function. RESULTS: Gastric emptying in cirrhotics showed no difference before or after paracentesis (median T1/2 108.0 min v. 117.9 min), but it was delayed significantly versus normal in both tests. There was no correlation with biochemical parameters, Child-Pugh score, or 13C-aminopyrine breath test results. Gastric half-emptying times of "full" peritoneal dialysis patients (median T1/2 103.1 min) were significantly higher than those of "empty" peritoneal dialysis patients (median T1/2 68.9 min) and asymptomatic volunteers (median T1/2 60.1 min). "Empty" peritoneal dialysis patients showed no gastroparesis. CONCLUSION: In alcoholic cirrhotic patients with ascites, gastric emptying of solids is delayed, independently of the volume of ascites. In peritoneal dialysis patients, gastric emptying was delayed when "full" and normalized after drainage of the dialysate.
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Esvaziamento Gástrico , Cirrose Hepática/fisiopatologia , Diálise Peritoneal , Adulto , Idoso , Testes Respiratórios , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , ParacenteseRESUMO
In four patients, two men and two women aged 73, 68, 84 and 72 years respectively, reversible cholestatic liver injury was seen 28-35 days after the start of treatment with amoxycillin-clavulanic acid (Augmentin). This rare complication of amoxycillin-clavulanic acid treatment is characterized by a relatively long latent period before the onset of symptoms or biochemical abnormalities, which makes early recognition difficult. The mechanism responsible for this idiosyncratic cholestasis is unknown.
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Colestase/induzido quimicamente , Quimioterapia Combinada/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Amoxicilina/efeitos adversos , Combinação Amoxicilina e Clavulanato de Potássio , Ácidos Clavulânicos/efeitos adversos , Feminino , Humanos , MasculinoRESUMO
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an alternatively spliced protein with two isoforms, TFPIα and TFPIß, which differ in their C-terminal structure and cellular localization. Detailed characterization of their inhibitory activity is needed to define potentially unique inhibitory roles in tissue factor (TF)-mediated thrombotic and inflammatory disease, and to understand how pharmaceuticals targeted to different structural regions of the TFPI isoforms alter hemostasis in hemophilia patients. METHODS: The TF inhibitory activity of TFPIß localized to the surface of CHO cells was compared with that of soluble TFPIα by the use of in vitro and in vivo assays. RESULTS: In TF-factor VIIa-mediated FXa generation assays, TFPIß was a slightly better inhibitor than TFPIα, which was approximately three-fold better than TFPI-160, a soluble, altered form of TFPI similar to TFPIß. In direct FXa inhibitory assays, TFPIß had an IC50 2.5-fold lower than that of TFPIα and 56-fold lower than that of TFPI-160. TFPIß inhibited TF-mediated CHO cell migration though Matrigel, whereas TFPIα and TFPI-160 were poor inhibitors, demonstrating that TFPIß effectively blocks TF-initiated signaling events during cellular migration through matrices that are not permeable to soluble forms of TFPI. Furthermore, TFPIß inhibited TF-dependent CHO cell infiltration into lung tissue following tail vein injection into SCID mice, and blocked the development of consumptive coagulopathy. CONCLUSIONS: TFPIß is a slightly better inhibitor of TF procoagulant activity than TFPIα. As a surface-associated protein, TFPIß is a much better inhibitor of TF-mediated cellular migration than soluble TFPIα, and may specifically act in the inhibition of TF-mediated signaling events on inflamed endothelium and/or monocytes.
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Lipoproteínas/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , HumanosRESUMO
BACKGROUND: Factor VII-activating protease (FSAP) is a serine protease that circulates in plasma in its inactive single-chain form and can be activated upon contact with dead cells. When activated by apoptotic cells, FSAP leads to the release of nucleosomes. The serpins C1-inhibitor and α(2) -antiplasmin are reported to be the major inhibitors of FSAP. However, regulation of FSAP activity by Kunitz-type inhibitors is not well studied. OBJECTIVES: To compare the inhibition of FSAP activity and FSAP-induced nucleosome release from apoptotic cells by tissue factor pathway inhibitor (TFPI) with that of C1-inhibitor and α(2) -antiplasmin. METHODS: Apoptotic cells were incubated with plasma or FSAP in presence of the inhibitor, and nucleosome release was analyzed with flow cytometry. Monoclonal antibodies against TFPI and altered forms of TFPI were used to investigate which domains of TFPI contribute to FSAP inhibition. RESULTS AND CONCLUSIONS: We show that TFPI abrogates FSAP activity and nucleosome release from apoptotic cells. TFPI is a much more efficient inhibitor than C1-inhibitor or α(2) -antiplasmin. The active site of K2 is required for inhibition of FSAP. A direct binding interaction between FSAP and the C-terminal domain of TFPI is also required for efficient inhibition. Inhibition of FSAP-induced nucleosome release by recombinant TFPI might, in part, explain the anti-inflammatory effects of recombinant TFPI infusion observed in animal and human sepsis.
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Apoptose , Lipoproteínas/farmacologia , Nucleossomos/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Anticorpos Monoclonais/metabolismo , Domínio Catalítico , Proteínas Inativadoras do Complemento 1/metabolismo , Proteína Inibidora do Complemento C1 , Relação Dose-Resposta a Droga , Ativação Enzimática , Citometria de Fluxo , Humanos , Células Jurkat , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Nucleossomos/enzimologia , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Inibidores de Serina Proteinase/imunologia , Inibidores de Serina Proteinase/metabolismo , alfa 2-Antiplasmina/metabolismoRESUMO
See also van den Boogaard FE, Brands X, Schultz MJ, Levi M, Roelofs JJTH, van 't Veer C, van der Poll T. Recombinant human tissue factor pathway inhibitor exerts anticoagulant, anti-inflammatory and antimicrobial effects in murine pneumococcal pneumonia. This issue, pp 122-32.