RESUMO
Fraction (F) II and FIII obtained by heparin-Sepharose after digestion of partially purified fibronectin (FN) with cathepsin D and F3, obtained like FIII but from untreated FN, exerted activity (arFN) on unfolded purified von Willebrand factor (vWF) that controls vWF multimer size. Our aim was to evaluate the arFN of F from commercial FN, commercial 30 kDa (with heparin affinity), 45 kDa (gelatin affinity) and 70 kDa FN fragments (gelatin and heparin affinity) and whole FN. The arFN was detected in FII, FIII, F2, F3, 30 kDa, 45 kDa and 70 kDa fragments. The least contaminated sample was the 30 kDa commercial fragment. Characterization studies of this sample revealed two bands: a blurred band of approximately 60 kDa and a sharp major band of 32 +/- 6 kDa. The 32 +/- 6 kDa band fragment failed to produce arFN because it was stronger than in F2 and FIII band fragments at the same position and with the same arFN. Our data suggest that a fragment of approximately 60 kDa that co-purified with FN, with affinity to heparin and gelatin, has the arFN that controls vWF multimer size.
Assuntos
Fibronectinas/química , Fragmentos de Peptídeos/farmacologia , Fator de von Willebrand/química , Biopolímeros , Eletroforese das Proteínas Sanguíneas , Catepsina D/metabolismo , Colágeno/metabolismo , Eletroforese em Gel de Ágar , Fibronectinas/isolamento & purificação , Humanos , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
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
Assuntos
Humanos , Metaloendopeptidases , Púrpura Trombocitopênica Trombótica , Fator de von Willebrand , Colágeno , Diálise , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/fisiopatologia , Metaloendopeptidases , Plasma , Púrpura Trombocitopênica Trombótica , Fator de von WillebrandRESUMO
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.
RESUMO
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)