RESUMO
BACKGROUND: Red blood cells (RBCs) contribute to hemostasis under blood-flow, and anemia might contribute to a hemorrhagic diathesis. The majority of current laboratory techniques to assess hemostasis do not consider the effects of RBCs. An assay to determine the role of RBCs in hemostasis could be beneficial for clinical management. OBJECTIVES: To investigate the influence of RBCs in hemostasis. METHODS: Hemostasis was investigated using a novel microchip flow-chamber system (T-TAS® ) in an anemic patient with von Willebrand disease. Subsequently, the effects of RBCs in total thrombus analysis system (T-TAS) were examined using reconstituted whole blood at various hematocrit levels. RESULTS: In vivo: When the patient was anemic and demonstrated persisted hemorrhagic symptoms despite the maintained adequate von Willebrand factor ristocetin cofactor activity levels, thrombus formation determined by T-TAS was delayed. However, transfusions of RBCs resolved bleeding symptom and, accordingly, the thrombus formation in T-TAS improved. In vitro: Thrombus formation determined by T-TAS at 1000 s-1 was dose-dependent on hematocrit (the time to reach 10 kPa (T10 ): 10.0 ± 0, 9.5 ± 1.4, 6.7 ± 2.4, 2.8 ± 1.6 min at hematocrits of 0%, 12.5%, 25% and 50%, respectively). Markedly defective thrombus formation (T10 >10 min) was confirmed at a hematocrit <25% at 2000 s-1 . CONCLUSION: Red blood cells play an essential role in hemostasis under high shear, and RBC transfusions could be effective for refractory bleeding in patients with anemia. T-TAS measurements appear to reflect the hemostatic consequences of diminished red cell numbers under blood-flow, and could provide a valuable means for monitoring patients.
Assuntos
Anemia/sangue , Anemia/complicações , Eritrócitos/metabolismo , Hemorragia/etiologia , Hemostasia , Resistência ao Cisalhamento , Anemia/diagnóstico , Testes de Coagulação Sanguínea , Pré-Escolar , Eritrócitos/patologia , Feminino , Humanos , Doenças de von Willebrand/sangue , Doenças de von Willebrand/complicações , Doenças de von Willebrand/diagnósticoRESUMO
INTRODUCTION: A recently developed method to assess comprehensive coagulation function, clot waveform analysis (CWA), accurately detect low levels (<1 IU/dL) of factor VIII activity (FVIII:C) in haemophilia A patients (HA-pts). Improvements are needed, however, to differentiate patients with very low from absent levels of FVIII:C. AIM: We attempted to optimize CWA using the coagulation analyser CS-2000i™ to distinguish between very low levels and absent FVIII:C in severe HA-pts. METHODS AND RESULTS: Activated partial thrombin time (aPTT)-based clot waveforms were determined in FVIII-deficient plasmas mixed with various amounts of recombinant FVIII. Clot times (CT) were shortened, and maximum coagulation velocity (|min1|) and acceleration (|min2|) were increased in FVIII dose-dependently at levels ranging from 0.25 to 100 IU/dL. The lowest level of FVIII:C detected was 0.25 IU/dL. Plasma samples from modestly severe (MS-HA; 0.5-<1.0 IU/dL), very severe (VS-HA; 0.25-<0.5 IU/dL), extremely severe (ES-HA; <0.25 IU/dL) and inhibitor-positive HA-pts (HA-inh) were examined. The CT was markedly prolonged in all instances but showed significant differences between the different groups insufficiently. The |min1| and |min2| in HA-inh were lower compared to the other groups (P<.05). A new parameter (slope-|min1|) reflecting average coagulation acceleration was derived. This index (median) was lower in HA-inh (0.0042) compared to ES-HA (0.0068) and VS-HA (0.011) with greater significant differences (P<.01), and an index of <.005 reflected the total absence of FVIII in the presence of inhibitor. CONCLUSION: The slope-|min1| parameter could provide a useful index for evaluating very low and absent levels of FVIII and/or the development of FVIII inhibitor in HA-pts.
Assuntos
Testes de Coagulação Sanguínea , Fator VIII , Hemofilia A/sangue , Hemofilia A/diagnóstico , Testes de Coagulação Sanguínea/instrumentação , Testes de Coagulação Sanguínea/métodos , Humanos , Tempo de Tromboplastina Parcial , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tempo de Coagulação do Sangue TotalRESUMO
The diagnosis of von Willebrand disease (VWD) is difficult due to the wide spectrum of clinical phenotypes associated with this disorder. We have analysed and characterized haemostatic function in VWD patients using a microchip-based flow chamber system. Microchips coated with either collagen [platelet (PL)-chip] or collagen/thromboplastin [atherome (AR)-chip] were used to evaluate platelet thrombus formation at 1000 s(-1) and fibrin-rich platelet thrombus formation at 240 s(-1) respectively. Blood samples from an asymptomatic patient with VWD type 1 [von Willebrand factor (VWF): RCo 3.2%; bleeding score (BS 2] displayed normal thrombus formation in both PL- and AR-chips, whereas blood from a symptomatic type 1 patient (VWF: RCo 14%, BS 9) had significantly delayed capillary occlusion. Nearly complete suppression of the flow pressure increase was observed in symptomatic patients with VWD type 2A (BS 13) and 2N (BS 27), whereas no flow pressure was found for the type 3 patient (BS 6). Fibrin-rich platelet thrombus formation was only weakly increased by the in vitro addition of factor VIII (FVIII) to blood samples from the type 3 patient, but was normalized by the addition of VWF/FVIII. The in vivo effects of treatment with desmopressin or VWF/FVIII for the symptomatic patients were analysed using two types of microchips. The PL-chip was highly sensitive for patients' VWF-mediated platelet functions, whereas the AR-chip allowed assessment of overall haemostatic ability, including sensitivity to both VWF and FVIII. The combined analysis with PL- and AR-chips may be potentially useful for the diagnosis of VWD based on clinical phenotypes, and for monitoring drug effects.
Assuntos
Desamino Arginina Vasopressina/uso terapêutico , Fator VIII/uso terapêutico , Hemostáticos/uso terapêutico , Doenças de von Willebrand/sangue , Fator de von Willebrand/uso terapêutico , Combinação de Medicamentos , Feminino , Hemostasia , HumanosAssuntos
Fatores de Coagulação Sanguínea/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemofilia A/imunologia , Hemorragia/complicações , Hemorragia/prevenção & controle , Fatores de Coagulação Sanguínea/administração & dosagem , Fatores de Coagulação Sanguínea/farmacologia , Criança , Hemofilia A/complicações , Humanos , Isoanticorpos/imunologia , Masculino , Falha de TratamentoRESUMO
Essentials Emicizumab mimics factor (F)VIIIa cofactor function, augments the intrinsic tenase activity. We assessed the emicizumab-driven hemostatic function in FXI-deficient plasmas. Emicizumab improved the coagulation potentials in severe FXI-deficient plasma. Emicizumab may provide a possibility for clinical application in patients with FXI deficiency. SUMMARY: Background Patients with factor (F)XI deficiency commonly present with markedly prolonged activated partial thromboplastin times (APTT), although bleeding phenotypes are heterogeneous. Emicizumab, a bispecific monoclonal antibody to FIX/FIXa and FX/FXa, mimics FVIIIa cofactor function on phospholipid (PL) surfaces. Antibody reactions were designed, therefore, to augment mechanisms during the propagation phase of blood coagulation. Aim To assess emicizumab-driven hemostatic function in FXI-deficient plasmas. Methods and Results Standard ellagic acid (Elg)/PL-based APTTs of different FXI-deficient plasmas (n = 13; FXI activity, < 1 IU dl-1 ) were markedly shortened dose dependently by the presence of emicizumab. To further analyze the effects of emicizumab, clot waveform analysis (CWA) in FXI-deficient plasmas with emicizumab, triggered by tissue factor (TF)/Elg demonstrated improvements in both clot times, reflecting the initiation phase, and coagulation velocity, which represents the propagation phase. Emicizumab also enhanced the TF/Elg-triggered thrombin generation in FXI-deficient plasmas dose-dependently although the degree of enhancement varied in individual cases. Thrombin generation with either FVII-deficient plasma or FIX-deficient plasma treated with anti-FXI antibody showed little or no increase by the co-presence of emicizumab, suggesting that the accelerated thrombin generation in FXI-deficient plasmas by emicizumab should depend on the FIXa-involved coagulation propagation initially triggered by FVIIa/TF. The ex vivo addition of emicizumab to whole blood from three patients with severe FXI deficiency demonstrated modest, dose-dependent improvements in Ca2+ -triggered thromboelastograms (NATEM mode). Conclusion Emicizumab appeared to improve coagulation function in severe FXI-deficient plasma, and might provide possibilities for clinical application in patients with FXI deficiency.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Coagulantes/farmacologia , Fator IX/antagonistas & inibidores , Fator X/antagonistas & inibidores , Inibidores do Fator Xa/farmacologia , Hemofilia B/tratamento farmacológico , Estudos de Casos e Controles , Fator IX/metabolismo , Fator X/metabolismo , Fator XIa/antagonistas & inibidores , Fator XIa/metabolismo , Fator Xa/metabolismo , Hemofilia B/sangue , Humanos , Tempo de Tromboplastina Parcial , Índice de Gravidade de Doença , Tromboelastografia , Trombina/metabolismoRESUMO
Precise measurements of factor VIII (FVIII) or factor IX (FIX) activity are believed to be essential for clinical management in haemophilia, although discrepancies between factor levels and clinical severity have been recognized. Clot wave form analysis has demonstrated that different wave form patterns may be evident in severe haemophilia A patients with levels of FVIII activity <1 IU dL(-1), and this might explain, in part, the phenotypic heterogeneity seen in these patients. In addition, the relatively new technique of computer-assisted thrombelastography (TEG), in which coagulation is initiated by tissue factor, has revealed a considerable degree of variability in different patients in the presence FVIII levels, which are sufficient to normalize TEG parameters. In contrast, a global thrombin generation test (TGT) has been proposed as a sensitive and reliable method for assessing overall clotting function in haemophilia patients. Several studies have demonstrated a significant correlation between TGT and FVIII/FIX levels, and these measurements also appear to correlate with the clinical phenotype. The TGT may be very useful, therefore, for evaluating overall haemostasis in different clinical situations, although substantial inter-assay and inter-individual variations have been reported. Both the TEG and TGT have been found to be particularly helpful for monitoring haemostatic therapy with bypassing agents or conventional FVIII or FIX concentrates in patients with inhibitors. These global tests enable the selection of appropriate therapeutic agents in individual circumstances and offer the opportunity to tailor the most effective haemostatic treatment even during severe bleeding or major surgery.
Assuntos
Fatores de Coagulação Sanguínea/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Hemofilia A/tratamento farmacológico , Fatores de Coagulação Sanguínea/fisiologia , Testes de Coagulação Sanguínea , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/tendências , Hemofilia A/sangue , Hemofilia A/diagnóstico , Hemostasia/efeitos dos fármacos , Hemostasia/fisiologia , Humanos , Sensibilidade e Especificidade , Tromboelastografia/instrumentaçãoRESUMO
UNLABELLED: Essentials von Willebrand factor (VWF) and factor VIII (FVIII) levels are modulated by age and ABO status. The effect of aging and ABO blood type on VWF and FVIII was assessed in 207 normal individuals. Aging and ABO blood type showed combined and bidirectional influences on VWF and FVIII levels. Aging and ABO blood type influence VWF levels through both secretion and clearance mechanisms. SUMMARY: Background The effect of aging and ABO blood type on plasma levels of von Willebrand factor (VWF) and factor VIII (FVIII) have been widely reported; however, a comprehensive analysis of their combined effect has not been performed and the mechanisms responsible for the age-related changes have not been determined. Objectives To assess the influence of aging and ABO blood type on VWF and FVIII levels, and to evaluate the contribution of VWF secretion and clearance to the age-related changes. Methods A cross-sectional observational study was performed in a cohort of 207 normal individuals, whose levels of VWF, FVIII, VWF propeptide (VWFpp), VWFpp/VWF:Ag ratio and blood type A antigen content on VWF (A-VWF) were quantified. Results Aging and ABO blood type exerted interrelated effects on VWF and FVIII plasma levels, because the age-related increase in both proteins was significantly higher in type non-O individuals (ß = 0.011 vs. 0.005). This increase with age in non-O subjects drove the differences between blood types in VWF levels, as the mean difference increased from 0.13 U/mL in the young to 0.57 U/mL in the old. Moreover, A-VWF was associated with both VWF antigen (ß = 0.29; 95% confidence interval [CI], 0.09, 0.50) and VWF clearance (ß = -0.15; 95% CI, -0.25, -0.06). We also documented an effect of ABO blood type on VWF secretion with aging, as old individuals with blood type non-O showed higher levels of VWFpp (mean difference 0.29 U/mL). Conclusions Aging and ABO blood type have an interrelated effect on VWF and FVIII levels, where the effect of one is significantly influenced by the presence of the other.
Assuntos
Sistema ABO de Grupos Sanguíneos , Envelhecimento , Fator VIII/análise , Fator de von Willebrand/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Tipagem e Reações Cruzadas Sanguíneas , Estudos de Coortes , Estudos Transversais , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The clinical phenotype of von Willebrand disease (VWD) is heterogeneous, and von Willebrand factor ristocetin cofactor activity (VWF:RCo) does not always reflect clinical severity, especially in VWD type 1. We have reported the potential of a microchip flow-chamber system (Total-Thrombus Formation Analysis System [T-TAS®]) for assessing physiologic hemostasis in VWD. Aim To evaluate the relationship between T-TAS, bleeding score (BS) and laboratory test results in type 1 VWD patients. METHODS: Microchips coated with collagen (platelet chip [PL-chip]) or collagen/thromboplastin (AR-chip) were used to assess platelet thrombus formation (PTF) at high shear rates or fibrin-rich PTF at low shear rates, respectively, in whole blood from 50 patients. The times needed for the flow pressure to increase by 10 kPa and 30 kPa (T10 and T30 ) from baseline were calculated from flow pressure curves. BS was determined by the use of a standardized questionnaire. RESULTS: PL-T10 values correlated with BS (R(2) ~ 0.45) better than VWF:RCo (R(2) ~ 0.36), irrespective of the flow rate, whereas AR-T10 showed only a weak correlation with BS (R(2) ~ 0.18). Patients with PL-T10 > 10 min or AR-T10 > 30 min had lower VWF levels and higher BS than those with PL-T10 ≤ 10 min or AR-T10 ≤ 30 min, and the greatest differences were observed with PL-T10. Clinical severity appeared to correlate best with PL-T10 > 8 min. BS was significantly higher in patients with VWF:RCo of < 10 IU dL(-1) than in those with VWF:RCo of 10 IU dL(-1) to < 25 IU dL(-1) and 25-40 IU dL(-1). In patients with VWF:RCo of < 10 IU dL(-1) , BS was significantly higher in those with PL-T10 > 8 min than in those with PL-T10 ≤ 8 min. CONCLUSION: T-TAS could be a useful technique for discriminating and predicting BS in VWD type 1 patients.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Doença de von Willebrand Tipo 1/sangue , Fator de von Willebrand/química , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Pré-Escolar , Colágeno/química , Feminino , Hemorragia , Hemostasia , Humanos , Lactente , Masculino , Microfluídica , Pessoa de Meia-Idade , Fenótipo , Pressão , Índice de Gravidade de Doença , Resistência ao Cisalhamento , Estresse Mecânico , Inquéritos e Questionários , Tromboplastina/química , Trombose , Adulto JovemRESUMO
BACKGROUND: The management of hemophilia A (HA) patients with inhibitors on bypassing therapy remains challenging. In particular, the monitoring of treatment is restricted by the limited reliability and lack of standardization of currently available methods to evaluate the physiological effects of various hemostatic agents. Accurate monitoring of these patients is particularly important in surgical situations. The recently developed comprehensive coagulation assays, including rotational thromboelastometry (ROTEM), may be useful in these circumstances. OBJECTIVE: We have attempted to establish a systematic monitoring protocol using ROTEM (NATEM triggered by CaCl2 ) to evaluate the choice and effectiveness of different bypassing agents in the perioperative period. METHODS AND RESULTS: The hemostatic effects of recombinant factor VIIa (rFVIIa) and activated prothrombin complex concentrates (aPCC) were determined using a three-step procedure (spike, preoperative and perioperative) in eight patients with HA inhibitor admitted for elective surgery and assessed for individually tailored therapy. The ROTEM parameters demonstrated similar improvement to approximately normal levels at each stage after treatment with rFVIIa. Results in the presence of aPCC showed a marked improvement in the spike data, although this appeared to be different from those in the preoperative and perioperative assessments. The information derived from the spike and preoperative findings provided a useful guide for establishing an effective dose of therapeutic material, and facilitated good hemostatic control during and after surgery in all cases. CONCLUSION: The findings suggest that this systematic analysis using ROTEM could provide a promising strategy for the use of bypassing therapy in HA patients with inhibitor.
Assuntos
Autoanticorpos/sangue , Fatores de Coagulação Sanguínea/uso terapêutico , Fator VIII/imunologia , Fator VIIa/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemostasia/efeitos dos fármacos , Hemostáticos/uso terapêutico , Assistência Perioperatória , Tromboelastografia/métodos , Adulto , Biomarcadores/sangue , Fatores de Coagulação Sanguínea/efeitos adversos , Criança , Pré-Escolar , Procedimentos Cirúrgicos Eletivos , Fator VIIa/efeitos adversos , Hemofilia A/sangue , Hemofilia A/diagnóstico , Hemofilia A/imunologia , Hemostáticos/efeitos adversos , Humanos , Masculino , Valor Preditivo dos Testes , Estudos Prospectivos , Proteínas Recombinantes/uso terapêutico , Índice de Gravidade de Doença , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: In mild hemophilia A (MHA) patients, the risk of inhibitor development is generally low, but some factor VIII (FVIII) gene missense mutations are associated with a higher inhibitor incidence. OBJECTIVE: To investigate the mechanism(s) of inhibitor development in MHA. METHODS AND RESULTS: A patient, HA78, with MHA with a novel P1809L missense mutation in the A3 domain, exhibited significant residual FVIII activity ( FVIII: C ~10 IU dL(-1) ), despite the development of an inhibitor (5.6 BU mL(-1) ). Purified HA78-IgG significantly depressed FVIII: C from normal plasma but not from patient's plasma without inhibitor, indicating that this IgG inhibited allogeneic but not autologous FVIII. The HA78-IgG blocked thrombin and FXa-catalyzed FVIII cleavage but had little effect on FVIII binding to von Willebrand factor and phospholipid. The IgG recognized a C2 epitope close or overlapping the previously described anti-C2 ESH8 epitope. Similarly, a recombinant FVIII-P1809L mutant was little inactivated by HA78-IgG. This mutant demonstrated ~3-fold lower binding affinities to von Willebrand factor and phospholipid compared with wild-type, while reactions with thrombin or FXa were not impaired. Reaction of FVIII-P1809L with the alternative anti-C2 ESH4 showed only an ~20% inhibition compared with wild-type FVIII but was similar to wild-type after incubation with ESH8. A surface plasmon resonance-based assay demonstrated that anti-C2 ESH4 bound to FVIII-P1809L with ~10(2) -fold lower affinity compared with ESH8. CONCLUSION: These results indicated that the P1809L mutation in A3 induced the conformational change in the FVIII molecule that hampered antigenic determinant(s) located in the C2 domain and might result in the inhibitor development.
Assuntos
Autoanticorpos/sangue , Coagulação Sanguínea/genética , Complemento C2/imunologia , Fator VIII/genética , Hemofilia A/genética , Mutação de Sentido Incorreto , Especificidade de Anticorpos , Testes de Coagulação Sanguínea , Reações Cruzadas , Análise Mutacional de DNA , Mapeamento de Epitopos , Epitopos , Fator VIII/química , Fator VIII/imunologia , Fator VIII/metabolismo , Predisposição Genética para Doença , Hemofilia A/sangue , Hemofilia A/diagnóstico , Humanos , Fenótipo , Estrutura Terciária de Proteína , Índice de Gravidade de Doença , Relação Estrutura-AtividadeRESUMO
The authors previously demonstrated that heparin immobilized surfaces showed excellent nonthrombogenic properties for extracorporeal membrane oxygenation experiments as long as 168 hr. The characteristics of the heparin immobilized surfaces include high heparin bioactivity and prevention of platelet adhesion and complement activation. However, it is not known whether the heparin immobilized surfaces would be effective for in vivo long-term implantation. Heparin bioactivity may be lost because of complete degradation or blocking of binding sites on heparin by adsorbed proteins. This study attempted to elucidate the in vivo long-term fate of heparin immobilized surfaces. The blood contacting surfaces of the ventricular assist device (VAD) made from polyurethane was modified with heparin immobilization and evaluated in a long-term sheep left VAD (LVAD) model for as long as 3 months. After removal of the VAD, heparin bioactivity was measured by Factor Xa assay. The blood contacting surfaces were analyzed with a scanning electron microscope, and the adsorbed proteins on the surfaces of the diaphragm were analyzed by SDS-PAGE and Western blotting. The thickness of adsorbed proteins on the surfaces also was measured by a confocal laser microscope. For the control ventricular assist devices, thrombus formation was observed within 1 month, whereas heparin immobilized VADs were able to operate thrombus free for periods as long as 3 months. The control surfaces demonstrated a thick adsorbed protein layer on thin surfaces, whereas heparin immobilized surfaces maintained thinner adsorbed proteins on thin surfaces. Anti Factor Xa activity of the heparinized surfaces disappeared after 15 days, but the surfaces remained nonthrombogenic even after heparin bioactivity was completely lost. The protein composition analyzed by SDS-PAGE showed an albumin dominant pattern on the heparinized surfaces. The band of 110 kD corresponding to C3b was detected only on the control surfaces, which possibly activated complement, and subsequently activated platelets and coagulation. Immunoblot showed degradation products of fibronectin and vitronectin on the control surfaces, which probably were promoted by surface generated protease, whereas the heparinized surfaces showed minimal degradation throughout the experimental periods. These results suggest that the heparin moiety has an ability to control adsorbed proteins, thereby inhibiting thrombus formation during in vivo long-term implantation.
Assuntos
Coração Auxiliar , Trombose/prevenção & controle , Adsorção , Animais , Complemento C3b/farmacocinética , Estudos de Avaliação como Assunto , Inibidores do Fator Xa , Coração Auxiliar/efeitos adversos , Heparina , Microscopia Eletrônica de Varredura , Desenho de Prótese , Proteínas/farmacocinética , Ovinos , Propriedades de Superfície , Fatores de TempoRESUMO
To evaluate the anti-tumour effect of recombinant feline interferon (rFeIFN) against canine neoplastic cells, the antiproliferation and anti-colony-forming activities of rFeIFN were investigated in vitro, using four cell lines derived from canine tumours; oral acanthomatous epulis (MCA-B1), mammary benign mixed tumour (MCM-B2), squamous cell carcinoma (CSCC), and malignant melanoma (CMC-1). The rFeIFN had a dose-dependent inhibitory effect on the cell growth and colony formation of all the cell lines, although the degree of inhibition was lower than that in the feline cell lines used as a positive control, and the sensitivity of the cells to rFeIFN differed.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Doenças do Cão/patologia , Interferons/farmacologia , Animais , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/veterinária , Doenças do Gato/patologia , Gatos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Transformação Celular Neoplásica/patologia , Cães , Relação Dose-Resposta a Droga , Feminino , Neoplasias Mamárias Animais/patologia , Melanoma/patologia , Melanoma/veterinária , Neoplasias Bucais/patologia , Neoplasias Bucais/veterinária , Tumor Filoide/patologia , Tumor Filoide/veterinária , Proteínas Recombinantes/farmacologia , Células Tumorais CultivadasRESUMO
Therapeutic effects of lividomycin (LVDM) were studied in 33 patients with respiratory infections including pneumonia, lung abscess, chronic bronchitis, etc. LVDM was intramuscularly administered at the dose of 1 or 2 g per day for consecutive 4 to 25 days. The results obtained are summarized below: 1. At the end of the first week of the treatment, rate of improvement in such parameters as cough, sputum, rales, fever and blood sedimentation rate were 69%, 56.7%, 60%, 79.2% and 70% respectively. Also, in 16% of the patients, abnormal shadow noted in X-ray film of the chest was disappeared and in 20% of the patients, size of the same was reduced during the first week of treatment. 2. Therapeutic effects of LVDM were evaluated synthetically and were graded as excellent, good, fair and ineffective. LVDM was effective in about 70 per cent of the patients, that is, excellent results were obtained in 4 patients, good in 12 patients, fair in 6 patients, and in 10 patients this antibiotic was ineffective. 3. In one patient with slight loss of high frequency perception was observed on the audiogram, but no other ototoxic effects such as subjective hearing loss, tinnitus, etc. In addition, no untoward effects on renal and hepatic function were observed. 4. The MIC values of LVDM for clinically isolated 20 strains of Pseudomonas aeruginosa were examined, using kanamycin for comparison. The MIC values of LVDM for many strains were superior to those of kanamycin. In view of the test results mentioned above, LVDM would appear to be useful medication for the treatment of some of respiratory infections.
Assuntos
Paromomicina/análogos & derivados , Paromomicina/uso terapêutico , Infecções Respiratórias/tratamento farmacológico , Doença Aguda , Adulto , Bronquiectasia/tratamento farmacológico , Bronquite/tratamento farmacológico , Doença Crônica , Feminino , Gentamicinas/farmacologia , Humanos , Técnicas In Vitro , Canamicina/farmacologia , Abscesso Pulmonar/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Paromomicina/farmacologia , Pneumonia/diagnóstico por imagem , Pneumonia/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Radiografia , Ribostamicina/farmacologia , Fatores de Tempo , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/diagnóstico por imagemRESUMO
BACKGROUND: Assays to determine the optimal hemostatic effects of bypass therapy in hemophilia A (HA) patients with inhibitors are difficult to compare. Clot waveform analysis (CWA), based on the continuous monitoring of routine coagulation parameters (prothrombin time/activated partial thromboplastin time), offers a useful method for assessing global clotting function. OBJECTIVES: To investigate the technique of CWA for the hemostatic monitoring of bypass therapy in HA patients with inhibitors. METHODS AND RESULTS: Ellagic acid (Elg), tissue factor (TF) or both (Elg/TF) were used as trigger reagents in CWA. The standard parameters - clot time (CT), maximum coagulation velocity (|min1|), and acceleration (|min2|) - were recorded. Optimal monitoring was defined as: (i) a significant difference in these parameters between plasma from HA patients with inhibitors and normal plasmas; and (ii) a significant improvement in these indices in HA patients with inhibitors after bypass therapy. Experiments in vitro demonstrated that there were significant differences between plasma from HA patients with inhibitors and normal plasma with various triggers, in the order Elg > Elg/TF >> TF. Addition of therapeutically achievable concentrations of bypassing agents, however, showed significant improvements in the different parameters only with Elg/TF, suggesting that this reagent provided the most appropriate assay. A total of 20 plasmas from HA patients with inhibitors in which bypassing agents were infused were evaluated ex vivo by Elg/TF CWA. The postinfusion parameters CT and |min2| reflected clinical effects, and were close to normal levels. Furthermore, Elg/TF CWA facilitated quantitative evaluation of perioperative hemostatic management of bypass therapy in HA patients with inhibitors. CONCLUSIONS: CWA is a promising method for the quantitative monitoring of bypass therapy during routine automated clotting assays with a modified trigger reagent comprising a well-balanced mixture of Elg and TF.
Assuntos
Fatores de Coagulação Sanguínea/química , Hemofilia A/sangue , Adolescente , Adulto , Coagulação Sanguínea , Testes de Coagulação Sanguínea/métodos , Criança , Fator VIII/química , Hemostasia , Humanos , Pessoa de Meia-Idade , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Tromboplastina/química , Adulto JovemRESUMO
We present a novel facility for micro-irradiation of living targets with ions from a 1.7 MV tandem accelerator. We show results using 1 MeV protons and 2 MeV He(2+). In contrast to common micro-irradiation facilities, which use electromagnetic or electrostatic focusing and specially designed vacuum windows, we employ a tapered glass capillary with a thin end window, made from polystyrene with a thickness of 1-2 µm, for ion focusing and extraction. The capillary is connected to a beamline tilted vertically by 45°, which allows for easy immersion of the extracted ions into liquid environment within a standard cell culture dish. An inverted microscope is used for simultaneously observing the samples as well as the capillary tip, while a stage-top incubator provides an appropriate environment for the samples. Furthermore, our setup allows to target volumes in cells within a µm(3) resolution, while monitoring the target in real time during and after irradiation.
Assuntos
Ambiente Controlado , Microtecnologia/instrumentação , Aceleradores de Partículas/instrumentação , Prótons , Sobrevivência Celular , Desenho de Equipamento , Células HeLa , HumanosRESUMO
BACKGROUND AND OBJECTIVES: Activated prothrombin complex concentrates (APCCs), utilized in bypassing therapy for hemophiliacs with inhibitor, contain factors (Fs) VII, FII, FIX and FX, and their active forms. A recent report has demonstrated that mixtures of APCC and FVIII potentiated thrombin generation, in vitro, in plasma from patients with severe hemophilia A, but the mechanism(s) involved remains unknown. RESULTS: APCC (0.05 U mL(-1) ) increased FVIII activity ~ 4-fold within 1 min in one-stage clotting assays, followed by a return to initial levels within 10 min. This reaction was dependent on the presence of tissue factor and phospholipid. Thrombin generation produced from APCC was ~ 3.5-fold greater in the presence of FVIII than that in its absence. SDS-PAGE analysis revealed that APCC sequentially proteolyzed the heavy chain of FVIII at Arg(372) and Arg(740) , followed by cleavage at Arg(336) . Proteolysis was prevented by FVIIa inhibitor, but not by hirudin, supporting the concept that APCC itself possessed the potential to activate FVIII in early coagulation phases, and that FVIIa in APCC contributed mainly to this reaction. APCC-mediated FVIII activation was unaffected by the addition of anti-FVIII inhibitor antibodies, irrespective of epitope specificity. Anti-C2 type 1 inhibitors, however, diminished the inactivation phase of the APCC reaction by inhibiting cleavage at Arg(336) . CONCLUSION: Small amounts of APCC, relative to the standard concentration used for clinical purposes, could activate FVIII directly, even in the presence of anti-FVIII antibodies. Combination therapy based on mixtures of APCC and FVIII could have significant beneficial implications for the treatment of hemophilia A patients with inhibitors.
Assuntos
Fator VIII/farmacologia , Hemostasia/efeitos dos fármacos , Protrombina/farmacologia , Autoanticorpos/imunologia , Misturas Complexas , Eletroforese em Gel de Poliacrilamida , Fator VIII/administração & dosagem , Fator VIII/química , Fator VIII/imunologia , Humanos , Proteólise , Protrombina/administração & dosagem , Protrombina/químicaRESUMO
Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 domain (residues 2228-2240) interacts with the FIXa Gla domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228-2240. The peptide 2228-2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 µM). This effect was significantly greater than that obtained by peptide 1804-1818 (IC50; ~180 µM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228-2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228-2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 domain (residues 2228-2240) with the FIXa Gla domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.
Assuntos
Coagulação Sanguínea , Fator IXa/metabolismo , Fator VIIIa/metabolismo , Fragmentos de Peptídeos/metabolismo , Cisteína Endopeptidases/metabolismo , Fator IXa/química , Fator VIIIa/química , Humanos , Cinética , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/química , Fosfolipídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Tempo de Protrombina , Proteínas Recombinantes/metabolismo , TromboelastografiaRESUMO
Mechanisms of inflammation and coagulation are linked through various pathways. Human neutrophil elastase (HNE), can bind to activated platelets, might be localised on platelet membranes that provide negatively-charged phospholipid essential for the optimum function of tenase complex. In this study, we examined the effect of HNE on factor (F)VIII. FVIII activity was rapidly diminished in the presence of HNE and was undetectable within 10 minutes. The inactivation rate was ~8-fold greater than that of activated protein C (APC). This time-dependent inactivation was moderately affected by von Willebrand factor. HNE proteolysed the heavy chain (HCh) of FVIII into two terminal products, A11-358 and A2375-708, by limited proteolysis at Val358, Val374, and Val708. Cleavage at Val708 was much slower than that at Val358 in the >90-kDa A1-A2-B compared to the 90-kDa A1-A2. The 80-kDa light chain (LCh) was proteolysed to 75-kDa product by cleavage at Val1670. HNE-catalysed FVIIIa inactivation was markedly slower than that of native FVIII (by ~25-fold), due to delayed cleavage at Val708 in FVIIIa. The inactivation rate mediated by HNE was ~8-fold lower than that by APC. Cleavages at Val358 and Val708 were regulated by the presence of LCh and HCh, respectively. In conclusion, HNE-catalysed FVIII inactivation was associated with the limited-proteolysis that led to A11-358, A2375-708, and A3-C1-C21671-2332, and subsequently to critical cleavage at Val708. HNE-related FVIII(a) reaction might play a role in inactivation of HNE-induced coagulation process, and appeared to depend on the amounts of inactivated FVIII and active FVIIIa which is predominantly resistant to HNE inactivation.