Visualization of von Willebrand Factor Multimers by Immunoenzymatic Stain Using Avidin-Biotin Peroxidase Complex

Aihara, Morio; Sawada, Yukimasa; Ueno, Kayo; Morimoto, Shigeo; Yoshida, Yutaka; Serres, Mark de; Cooper, Herbert A.; Wagner, Robert H.

doi:10.1055/s-0038-1661533

Cited by 52 publications

(42 citation statements)

References 16 publications

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“…Owing to this second dimension, however, the triplet structure shows a higher resolution than in the commonly used SDS-agarose gel electrophoresis. The triplet structure consists of a more prominent intermediate band, a faster migrating band, and a slower migrating satellite band [26][27][28]. Some of these bands may be missing or may migrate with an aberrant mobility in vWD variants [29][30][31][32], suggesting that the complex multimeric structure of plasma vWF may have pathophysiological significance.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2011

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Blue native electrophoresis (BNE) was applied to analyze the von Willebrand factor (vWF) multimers in their native state and to present a methodology to perform 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. Thereby, we obtained a pattern in which each protein sub‐unit from the first dimension dissociates 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 additional 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 potential enables us to compare the triplet structure from platelet and plasmatic vWF, 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 disorder, and for the classification of von Willebrand's disease subtypes.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2011

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“…VWF antigen levels in citrated plasma were measured by a Rellplate electroimmunodiffusion kit (American Diagnostics, Seattle, WA, U.S.A.). The multimeric distribution of VWF was analyzed by separating the multimers by SDS-1.5% agarose gel electrophoresis as described (31,32), and determining the relative proportions of low (1-to 5-mers), intermediate (6-to 10-mers), and large (Ͼ10-mers) multimers of VWF by densitometry.…”

Section: Studymentioning

confidence: 99%

The Relationship of von Willebrand Factor Binding to Activated Platelets from Healthy Neonates and Adults

et al. 2003

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“…Again after several washes, the membrane is ''developed'' using a specific substrate that is converted by the conjugated enzyme to elicit a color signal. Numerous methods [13,16,18,[24][25][26][27][28][29] (Table I) have been published using vari- ous primary antibodies, secondary conjugated antibodies, and substrates.…”

Section: Detection Of Protein (Visualization)mentioning

confidence: 99%

Analysis of von Willebrand factor structure by multimer analysis

Ledford‐Kraemer¹

2010

American J Hematol

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Analysis of von Willebrand factor (VWF) structure is achieved by performing a highly specialized procedure, VWF multimer analysis. The test is reserved for the reference or specialized laboratory environment. The assay is qualitative (though under some circumstances multimers may be quantified) in that it assesses the overall size distribution of VWF multimers as well as their individual internal structure. The test is used predominantly to type or subtype von Willebrand disease. The analysis of VWF multimers generally consists of four steps: (1) electrophoresis of plasma in an agarose gel, (2) either gel fixation or transfer of the electrophoretic protein product to a membrane, (3) immunodetection of the protein, and (4) evaluation of the protein in the gel or membrane. The assay is complex, time consuming, requires specialized equipment and technical expertise, and is not standardized. Am. J. Hematol. 85:510-514, 2010. V

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Visualization of von Willebrand Factor Multimers by Immunoenzymatic Stain Using Avidin-Biotin Peroxidase Complex

Cited by 52 publications

References 16 publications

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

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

The Relationship of von Willebrand Factor Binding to Activated Platelets from Healthy Neonates and Adults

Analysis of von Willebrand factor structure by multimer analysis

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