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 VIII/von Willebrand factor (FVIII/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 FVIII/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 FVIII/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 approximately 50% of the vWF function.
D000478:Alkylation; D001779:Blood Coagulation Factors / Q000502:physiology; D001792:Blood Platelets / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D005169:Factor VIII / Q000502:physiology; D006801:Humans; D046911:Macromolecular Substances; D008623:Mercaptoethanol / Q000494:pharmacology; D008854:Microscopy, Electron; D008970:Molecular Weight; D010974:Platelet Aggregation; D011487:Protein Conformation; D012310:Ristocetin / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000502:physiology
D001779:Blood Coagulation Factors; D046911:Macromolecular Substances; D014841:von Willebrand Factor; D012310:Ristocetin; D008623:Mercaptoethanol; D005169:Factor VIII; D004229:Dithiothreitol