Native multimer analysis of plasma and platelet von Willebrand factor compared to denaturing separation: Implication for the interpretation of satellite bands

Abstract

Blue native electrophoresis (BNE) was used to analyze the von Willebrand factor (vWF) multimers in their native state and to present a methodology for performing blue native electrophoresis on human plasma proteins which has not been done before. The major difference between this method and the commonly used SDS-agarose gel electrophoresis is the lack of satellite bands in the high-resolution native gel. To further analyze this phenomenon, a second dimension was performed under denaturing conditions. This way we obtained a pattern in which each protein sub-unit from the first dimension dissociated into three distinct sub-bands. These bands confirm the triplet structure, which consists of an intermediate band and two satellite bands. By introducing the second dimension, our novel method separates the triplet structure into a higher resolution than the commonly used SDS-agarose gel electrophoresis does. This helps considerably in the classification of ambiguous von Willebrand's disease subtypes. In addition, our method has the advantage of being able to resolve the triplet structure of platelet vWF multimers, which has not been identified previously through conventional SDS-agarose electrophoresis multimer analysis. This enables us to compare the triplet structures of platelet and plasmatic vWFs, and may help to find out whether structural abnormalities concern the vWF molecule in the platelet itself, or whether they are due to the physiological processing of vWF shed into circulation. Owing to its resolution and sensitivity, this native separation technique offers a promising tool for the analysis and detection of von Willebrand disorders, and for the classification of von Willebrand's disease subtypes.