RESUMO
La disfunción ADAMTS13 ha sido implicada en la patogénesis de la púrpura trombocitopénica trombótica (PTT). Este desorden ocurre con mayor frecuencia en mujeres y, en el 13 por ciento de ellas, se asocia al embarazo. Sin embargo, hay poca información sobre el comportamiento de la proteasa en embarazo normal. Estudiamos el factor de de von Willebrand y la actividad ADAMTS13 en mujeres sanas no embarazadas, embarazadas y puerperio. Se incluyeron a cincuenta y cinco mujeres no embarazadas, donantes normales del banco de sangre, que no tomaban píldoras anticonceptivas, como controles. Se incluyeron 270 embarazadas y puérperas normales. La actividad ADAMTS13 disminuyó progresivamente a partir del período de 12-16 semanas hasta el final del puerperio temprano (media el 52 por ciento, rango 22-89, p < 0,0001), con un aumento leve posterior. Los niveles fueron levemente menores en nulíparas que en mujeres con paridad previa (65 por ciento vs 83 por ciento, p = 0,0003), y primíparas que en multiparas entre 6-11 semanas hasta 17-23 semanas del embarazo (69 por ciento vs. 80 por ciento, p = 0,005). Aunque en todas las mujeres los níveles de proteasa fueron iguales en los diferentes grupos sanguíneos, las mujeres no embarazadas del grupo sanguíneo O demostraron una media mayor de actividad de ADAMTS13 que el no-O (78 por ciento vs 69 por ciento, p = 0,064). Nuestros resultados sugieren que los cambios de actividad de ADAMTS13, durante el embarazo y puerperio, podrían hacer que el final del embarazo fuera un período más vulnerable para el desarrollo de microangiopatía trombótica.
Assuntos
Humanos , Adulto , Feminino , Gravidez , Proteínas ADAM , Metaloendopeptidases/sangue , Estudos de Coortes , Estudos Transversais , Fator de von Willebrand/biossíntese , Imunoeletroforese , Contagem de Plaquetas , Período Pós-Parto , Primeiro Trimestre da Gravidez , Púrpura Trombocitopênica Trombótica/etiologia , Púrpura Trombocitopênica Trombótica/patologia , Valores de Referência , Fatores de TempoRESUMO
ADAMTS13 dysfunction has been involved in the pathogenesis of Thrombotic Thrombocytopenic Purpura. This disorder occurs more frequently in women and, in 13% of them, is associated with pregnancy. However, there is little information on the protease behaviour in normal pregnancy. We studied von Willebrand factor and ADAMTS13 activity changes in normal non-pregnant, pregnant and post-delivery women. Fifty-five non-pregnant women, normal blood bank donors, who were not taking contraceptive pills were included as controls. A prospective cross-sectional study of 270 normal pregnant and post-delivery women was carried out. ADAMTS13 activity decreased progressively as from the period of 12-16 weeks up to the end of early puerperium (mean 52%, range 22-89, p < 0.0001), to increase slightly thereafter. Nulliparous presented mildly lower levels of ADAMTS13 activity than parous women (65% vs. 83 %, p = 0.0003), and primigravidae than multigravidae between 6-11 weeks up to 17-23 weeks of pregnancy (69% vs. 80%, p = 0.005). Although in all women the protease levels were the same by blood groups, the O blood group non-pregnant women showed a higher mean of ADAMTS13 activity than those non-O (78% vs. 69%, p = 0.064). Our results suggest that the changing levels of protease activity during pregnancy and puerperium, induced by unidentified mechanisms, could render the peripartum time more vulnerable to developed thrombotic microangiopathies.
Assuntos
Metaloendopeptidases/sangue , Proteínas ADAM , Proteína ADAMTS13 , Adulto , Estudos de Coortes , Estudos Transversais , Feminino , Humanos , Imunoeletroforese , Contagem de Plaquetas , Período Pós-Parto , Gravidez , Trimestres da Gravidez , Valores de Referência , Fatores de Tempo , Fator de von Willebrand/biossínteseRESUMO
The von Willebrand factor cleaving protease (VWFCP) modulates the von Willebrand factor (VWF) multimeric size in normal plasma. VWFCP activity levels are decreased in different physiological and pathologic situations. Different techniques have been developed to unfold the purified VWF (perfusion at high shear rate, dialysis against urea in nitrocellulose filters), to detect the VWFCP activity on it (multimeric analysis of VWF, collagen binding to VWF assay) and to use the patient plasma both as the source of the enzyme and substrate. In this paper we compared the above mentioned methods with new ones: normal plasma dialyzed on membranes instead of purified VWF, dialysis of the samples against urea in tubing instead of nitrocellulose filters, and sonicated plasma to remove the endogenous VWF. The perfusion assay and detection by multimeric analysis showed a limit of detection (25%) of VWFCP activity. Dialysis against urea in both supports and detection by multimeric analysis, showed a better limit of detection (3%), but the recovery of the samples was not as efficient in nitrocellulose filters as it was in tubing. The detection by collagen binding to VWF has more advantages because it allows to analyze more samples than the multimeric analysis does in the same assay. The dialysis of plasma by membranes to obtain the source of exogenous VWF requires no complex equipment. The method, which uses patient plasma as the source of the enzyme and substrate, was inapplicable in our experience because the values could not be interpolated in the reference curve.
Assuntos
Metaloendopeptidases/isolamento & purificação , Púrpura Trombocitopênica Trombótica/sangue , Fator de von Willebrand/química , Proteínas ADAM , Proteína ADAMTS13 , Colágeno/metabolismo , Diálise , Síndrome Hemolítico-Urêmica/sangue , Humanos , Metaloendopeptidases/sangue , Sensibilidade e EspecificidadeRESUMO
The von Willebrand factor cleaving protease (VWFCP) modulates the von Willebrand factor (VWF) multimeric size in normal plasma. VWFCP activity levels are decreased in different physiological and pathologic situations. Different techniques have been developed to unfold the purified VWF (perfusion at high shear rate, dialysis against urea in nitrocellulose filters), to detect the VWFCP activity on it (multimeric analysis of VWF, collagen binding to VWF assay) and to use the patient plasma both as the source of the enzyme and substrate. In this paper we compared the above mentioned methods with new ones: normal plasma dialyzed on membranes instead of purified VWF, dialysis of the samples against urea in tubing instead of nitrocellulose filters, and sonicated plasma to remove the endogenous VWF. The perfusion assay and detection by multimeric analysis showed a limit of detection (25%) of VWFCP activity. Dialysis against urea in both supports and detection by multimeric analysis, showed a better limit of detection (3%), but the recovery of the samples was not as efficient in nitrocellulose filters as it was in tubing. The detection by collagen binding to VWF has more advantages because it allows to analyze more samples than the multimeric analysis does in the same assay. The dialysis of plasma by membranes to obtain the source of exogenous VWF requires no complex equipment. The method, which uses patient plasma as the source of the enzyme and substrate, was inapplicable in our experience because the values could not be interpolated in the reference curve (AU)
Assuntos
Humanos , Fator de von Willebrand , Metaloendopeptidases/metabolismo , Púrpura Trombocitopênica Trombótica/fisiopatologia , Metaloendopeptidases/sangue , Púrpura Trombocitopênica Trombótica/metabolismo , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/fisiopatologia , Diálise , Fator de von Willebrand/isolamento & purificação , Fator de von Willebrand/metabolismo , Plasma/enzimologia , Colágeno/metabolismoRESUMO
Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement (C)-dependent intravascular hemolysis. The authors studied the mechanism of induction of C-induced hemolysis. Purified Loxosceles toxins rendered human erythrocytes susceptible to lysis by human C but did not have an effect on the E-bound C-regulators DAF, CR1, or CD59. However, incubation with venom toxins caused cleavage of glycophorin from the erythrocyte (E) surface, facilitating C activation and hemolysis. The results suggest that glycophorin is an important factor in the protection of E against homologous C. Cleavage of glycophorin (GP) A, GPB, and GPC occurred at sites close to the membrane but could not be accomplished using purified GPA and purified toxins, demonstrating that cleavage was not an effect of a direct proteolytic action of the Loxosceles toxins on the glycophorins. Inhibition of the cleavage of glycophorins induced by Loxosceles venom was achieved with 1,10-phenanthroline. The authors propose that the sphingomyelinase activity of the toxins induces activation of an endogenous metalloproteinase, which then cleaves glycophorins. They observed the transfer of C-dependent hemolysis to other cells, suggesting that the Loxosceles toxins can act on multiple cells. This observation can explain the extent of hemolysis observed in patients after envenomation. Identification of the mechanism of induction of susceptibility to C-mediated lysis after Loxosceles envenomation opens up the possibility of the development of an effective therapeutic strategy. (Blood. 2000;95:683-691)
Assuntos
Proteínas do Sistema Complemento/fisiologia , Membrana Eritrocítica/fisiologia , Eritrócitos/fisiologia , Glicoforinas/efeitos dos fármacos , Hemólise , Metaloendopeptidases/sangue , Diester Fosfórico Hidrolases/farmacologia , Venenos de Aranha/farmacologia , Animais , Ativação Enzimática , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Glicoforinas/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Células Jurkat , Células K562 , Metaloendopeptidases/efeitos dos fármacos , Neuraminidase/farmacologia , Diester Fosfórico Hidrolases/isolamento & purificação , Inibidores de Proteases/farmacologia , Venenos de Aranha/isolamento & purificação , Aranhas , Células U937RESUMO
Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement (C)-dependent intravascular hemolysis. The authors studied the mechanism of induction of C-induced hemolysis. Purified Loxosceles toxins rendered human erythrocytes susceptible to lysis by human C but did not have an effect on the E-bound C-regulators DAF, CR1, or CD59. However, incubation with venom toxins caused cleavage of glycophorin from the erythrocyte (E) surface, facilitating C activation and hemolysis. The results suggest that glycophorin is an important factor in the protection of E against homologous C. Cleavage of glycophorin (GP) A, GPB, and GPC occurred at sites close to the membrane but could not be accomplished using purified GPA and purified toxins, demonstrating that cleavage was not an effect of a direct proteolytic action of theLoxosceles toxins on the glycophorins. Inhibition of the cleavage of glycophorins induced by Loxosceles venom was achieved with 1,10-phenanthroline. The authors propose that the sphingomyelinase activity of the toxins induces activation of an endogenous metalloproteinase, which then cleaves glycophorins. They observed the transfer of C-dependent hemolysis to other cells, suggesting that the Loxosceles toxins can act on multiple cells. This observation can explain the extent of hemolysis observed in patients after envenomation. Identification of the mechanism of induction of susceptibility to C-mediated lysis afterLoxosceles envenomation opens up the possibility of the development of an effective therapeutic strategy.