DISULFIDE BONDS REQUIRED TO ASSEMBLE FUNCTIONAL VON-WILLEBRAND-FACTORMULTIMERS

The hemostatic functions of human von Willebrand Factor (VWF) depend o n the normal assembly of disulfide-linked multimers from similar to 25 0-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 plasm a vWF and recombinant vWF fragments indicate that 1 or more of the Cys residues at position 459, 462, and 464 form intersubunit disulfide bo nds. No evidence has been reported that vWF multimer formation involve s additional intersubunit bonds. To probe the disulfide bond requireme nts for multimer formation, mutant vWF proteins were expressed in whic h all 3 Cys residues at positions 459, 462, and 464 were changed to ei ther Gly or Ala. Surprisingly, none of these cysteines appears to be n ecessary for efficient multimer assembly. Furthermore, recombinant vWF with Gly or Ala at all three positions induces platelet aggregation i n the presence of ristocetin and binds to platelet glycoprotein Ib, fa ctor VIII, and collagen in a manner similar to wild-type recombinant v WF. These results suggest that other intersubunit disulfide bonds must exist. Direct evidence for such a bond was obtained by characterizati on of tryptic fragments of vWF. By Edman degradation, amino acid compo sition, and mass spectrometry, a disulfide bond was demonstrated betwe en Cys(379) residues of adjacent vWF subunits, Thus, intersubunit disu lfide bonds involving Cys(379) and 1 or more of the Cys residues at po sitions 459, 462, and 464 connect the NH2-terminal ends of the vWF sub units in a parallel orientation.