Electron microscopy of human factor VIII/von willebrand glycoprotein: Effect of reducing reagents on structure and function
The structure of native and progressively reduced human factor Vlll/von Willebrand factor (FVlll/vWF) was examined by electron microscopy and SDS gel electrophoresis and then correlated with its biological activities. Highly resolved electron micrographs of well-spaced, rotary-shadowed FVlII/vWF molecules showed their structure to consist of a very flexible filament that contains irregularly spaced small nodules. Filaments ranged from 50 to 1,150 nm with a mean length of 478 nm and lacked fixed, large globular domains as seen in fibrinogen and IgM. A population of multimeric FVlII/vWF species ranging in molecular weight from 1 to 5 million daltons and differing in size alternately by one and two subunits was observed on SDS-2% polyacrylamide-0.5% agarose gel electrophoresis. With progressive reduction of disulfide bonds by dithiothreitol (DTT), the electron microscopic size of FVIII/vWF decreased in parallel with increased electrophoretic mobility on SDS-agarose gels; between 0.1 and 0.5 mM DTT its structure changed from predominantly fibrillar species to large nodular forms. A 50% loss of vWF specific activity and FVIII procoagulant activity occurred at 0.4 mM DTT and 1 mM DTT, respectively, corresponding to the reduction of 4 and 12 disulfide bonds of the 62 disulfides per 200,000-dalton subunit. We conclude that reduction of a few critical disulfide bonds results in a major structural change by electron microscopy and a concomitant loss of ~50% of the vWF function. © 1982, Rockefeller University Press., All rights reserved.
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- Developmental Biology
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Citation
Published In
DOI
EISSN
ISSN
Publication Date
Volume
Issue
Start / End Page
Related Subject Headings
- Developmental Biology
- 32 Biomedical and clinical sciences
- 31 Biological sciences
- 11 Medical and Health Sciences
- 06 Biological Sciences