Disulfide bonds required to assemble functional von Willebrand factor multimers

• Published in: The Journal of biological chemistry 1994-03, Vol.269 (9), p.6753-6758
• Main Authors: ZHENGYU DONG, THOMA, R. S, CRIMMINS, D. L, MCCOURT, D. W, TULEY, E. A, SADLER, J. E
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Blood coagulation. Blood cells; Blood Platelets - drug effects; Blood Platelets - metabolism; Cell Line; Collagen - metabolism; Crotalid Venoms - pharmacology; Disulfides - metabolism; DNA, Complementary - metabolism; Electrophoresis, Polyacrylamide Gel; Factor VIII - metabolism; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods, hemostasis, fibrinolysis; Humans; Kinetics; Macromolecular Substances; Models, Structural; Molecular and cellular biology; Molecular Sequence Data; Molecular Weight; Oligodeoxyribonucleotides; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Secondary; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Restriction Mapping; Transfection; von Willebrand Factor - biosynthesis; von Willebrand Factor - chemistry; von Willebrand Factor - isolation & purification
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37439-2

The hemostatic functions of human von Willebrand Factor (vWF) depend on the normal assembly of disulfide-linked multimers from approximately 250-kDa subunits. Subunits initially form dimers through disulfide bonds near the COOH terminus. Dimers then form multimers through disulfide bonds near the NH2 terminus of each subunit. Previous studies of plasma vWF and recombinant vWF fragments indicate that 1 or more of the Cys residues at position 459, 462, and 464 form intersubunit disulfide bonds. No evidence has been reported that vWF multimer formation involves additional intersubunit bonds. To probe the disulfide bond requirements for multimer formation, mutant vWF proteins were expressed in which all 3 Cys residues at positions 459, 462, and 464 were changed to either Gly or Ala. Surprisingly, none of these cysteines appears to be necessary for efficient multimer assembly. Furthermore, recombinant vWF with Gly or Ala at all three positions induces platelet aggregation in the presence of ristocetin and binds to platelet glycoprotein Ib, factor VIII, and collagen in a manner similar to wild-type recombinant vWF. These results suggest that other intersubunit disulfide bonds must exist. Direct evidence for such a bond was obtained by characterization of tryptic fragments of vWF. By Edman degradation, amino acid composition, and mass spectrometry, a disulfide bond was demonstrated between Cys379 residues of adjacent vWF subunits. Thus, intersubunit disulfide bonds involving Cys379 and 1 or more of the Cys residues at positions 459, 462, and 464 connect the NH2-terminal ends of the vWF subunits in a parallel orientation.