Triplet structure of human von Willebrand factor.
Human von Willebrand factor (hp-vWF) is a high-molecular- mass protein found in plasma as a series of multimers. It consists of subunits comprising 2050 amino acids linked by disulphide bonds into multimers of various size ranging in molecular mass up to greater than 10000kDa. Partial proteolysis at position Tyr842-Mer843 of the subunit [Dent et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6306-6310] by a vWF-specific protease [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503-7507] results in the generation of an N-terminal and a C-terminal fragment and the appearance of hp-vWF triplet bands. It has been suggested [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503-7507] that (i) the intermediate triplet band of the primary dimer represents a dimer of two C-terminal fragments, (ii) the slower migrating satellite band of the primary dimer represents an asymmetric structure composed of a mature subunit to which one N-terminal and one C-terminal fragment are linked by disulphide bonds, and (iii) the faster migrating satellite band of the primary dimer contains two N-terminal fragments. Here we used recombinant vWF (r-vWF) for structural analysis of hp-vWF multimers. r-vWF exhibited no proteolytic degradation and all multimers contained mature subunits. High-resolution agarose-gel electrophoresis and two-dimensional electrophoresis demonstrated that (i) r-vWF multimers and hp-vWF intermediate triplet bands exhibited identical molecular mass and electrophoretic mobilities, (ii) the faster and slower migrating satellite bands of hp-vWF differ by less than the molecular mass of one subunit from the corresponding intermediate triplet band, and (iii) the triplet bands of hp-vWF are composed of mature and degraded subunits. The results support a structural model of hp-vWF triplet bands according to which the intermediate triplet bands represent multiple numbers of symmetric and/or asymmetric dimers, the slower migrating satellite bands have one extra N-terminal fragment, and the faster migrating satellite band lacks one N-terminal fragment respectively in comparison with the corresponding intermediate triplet band.
Fischer
B E
BE
IMMUNO AG, Biomedical Research Center, Uferstrasse. 15, A-2304 Orth/Donau, Austria.
Thomas
K B
KB
Schlokat
U
U
Dorner
F
F
D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D014841:von Willebrand Factor / Q000737:chemistry*
D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014841:von Willebrand Factor; D010450:Endopeptidases