Oligomerization of the VWF C2 domain is mediated by the Cys2431-Cys2453 and Cys2451-Cys2468 disulfides/dithiols. (A) The VWF C1-C3 fragment was expressed in mammalian HEK cells and purified from the conditioned medium. The fragment was resolved on SDS-PAGE and blotted with anti-VWF polyclonal Abs. The fragment was present as monomers, dimers, and trimers. (B) The Myc-tagged VWF C2 fragment was expressed in mammalian HEK cells and purified from the conditioned medium. The fragments were resolved on SDS-PAGE and blotted with anti-Myc Abs. The C2 domain was present as monomers, dimers, trimers, tetramers, and higher-order oligomers. The C2 domain multimers were disulfide-linked, because they resolved to monomers on incubation with DTT. (C) Wild-type or mutants of the Cys2431-Cys2453 and Cys2451-Cys2468 disulfide bonds of the Myc-tagged C2 fragment were expressed in mammalian HEK cells, purified from the conditioned medium, resolved on SDS-PAGE, and blotted with anti-Myc Abs. The wild-type C2 fragment was present as monomers, dimers, trimers, and higher-order oligomers. The C2431A and C2431A,C2453A C2 mutants were only present as monomers, the Cys2451A mutant only as monomers and dimers, whereas the C2453A and C2468A mutants demonstrated enhanced oligomerization.