Lateral self-association of VWF involves the Cys2431-Cys2453 disulfide/dithiol in the C2 domain

VWF is a plasma protein that binds platelets to an injured vascular wall during thrombosis. When exposed to the shear forces found in flowing blood, VWF molecules undergo lateral self-association that results in a meshwork of VWF fibers. Fiber formation has been shown to involve thiol/disulfide exchange between VWF molecules. A C-terminal fragment of VWF was expressed in mammalian cells and examined for unpaired cysteine thiols using tandem mass spectrometry (MS). The VWF C2 domain Cys2431-Cys2453 disulfide bond was shown to be reduced in approximately 75% of the molecules. Fragments containing all 3 C domains or just the C2 domain formed monomers, dimers, and higher-order oligomers when expressed in mammalian cells. Mutagenesis studies showed that both the Cys2431-Cys2453 and nearby Cys2451-Cys2468 disulfide bonds were involved in oligomer formation. Our present findings imply that lateral VWF dimers form when a Cys2431 thiolate anion attacks the Cys2431 sulfur atom of the Cys2431-Cys2453 disulfide bond of another VWF molecule, whereas the Cys2451-Cys2468 disulfide/dithiol mediates formation of trimers and higher-order oligomers. These observations provide the basis for exploring defects in lateral VWF association in patients with unexplained hemorrhage or thrombosis.