Solution studies of the quaternary structure and assembly of human von Willebrand factor.
The reversible association of protomers of von Willebrand protein (vWF) was studied in order to analyze the forces and mechanism of vWF polymer assembly. At concentrations of vWF found in plasma (approximately 16 micrograms/mL), disulfide bond reduction with 50 mM 2-mercaptoethanol (2-ME) markedly reduced both vWF activity, as measured by ristocetin-dependent platelet agglutination, and average polymer size (Rh, the mean hydrodynamic radius) in solution, as determined by quasi-elastic light scattering (QLS) and by gel filtration chromatography. With increasing vWF concentration, activity and Rh increased despite reduction of interprotomer disulfide bonds. Changes in temperature after 2-ME treatment produced reversible changes in activity and Rh. Varying the total vWF concentration at any given temperature after 2-ME treatment changed Rh in a consistent and predictable fashion, so that estimates of the dissociation constant for vWF protomer-polymer equilibrium were obtained: Kd5 degrees C = 0.77 micrograms/mL, Kd25 degrees C = 2.4 micrograms/mL, and Kd37 degrees C = 7.7 micrograms/mL, where under the conditions of reduction presented here, the basic protomer of vWF is a dimer. Increasing ionic strength after 2-ME treatment with 1 M KCl did not change Rh, while approximately 100 microM sodium dodecyl sulfate (SDS) or approximately 300 microM sodium deoxycholate (DOC) reduced both Rh and activity compared with those of unreduced polymer. These data show that disulfide bonds are necessary to maintain vWF polymer size and activity at plasma concentrations but that noncovalent forces of association can maintain vWF polymer size and activity at higher concentrations. These forces of association may be important for polymer assembly during intracellular synthesis of vWF.
D004220:Disulfides; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008623:Mercaptoethanol / Q000494:pharmacology; D010973:Platelet Adhesiveness; D011487:Protein Conformation; D012996:Solutions; D013816:Thermodynamics; D014841:von Willebrand Factor / Q000378:metabolism*
D004220:Disulfides; D046911:Macromolecular Substances; D012996:Solutions; D014841:von Willebrand Factor; D008623:Mercaptoethanol