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Abstract
Von Willebrand Factor (VWF) is a multimeric plasma protein responsible for tethering platelets to an injured vascular wall and chaperoning coagulation Factor VIII. When exposed to shear stress VWF multimers can undergo a lateral (homotypic) self-association which results in the formation of a mesh of VWF fibres that platelets readily bind to. Plasma VWF contains free thiols and lateral self-association can be inhibited by thiol alkylating agents, implying that this process involves a thiol-disulphide exchange between VWF multimers.
Recombinant C-terminal VWF fragments were expressed in a mammalian cell system and unpaired cysteine residues characterized using tandem mass spectrometry. Wild-type VWF fragments containing the C2 domain formed monomers, dimer and higher order oligomers when expressed in this system. The Cys2431-Cys2453 disulphide bond in the C2 domain of VWF was found to be reduced in approximately 75% of the molecules. From mutagenesis studies, both the Cys2431-Cys2453 and nearby Cys2451-Cys2468 disulphide bonds were found to play a role in oligomerisation.
The findings imply that lateral VWF dimers form when the Cys2431 thiolate anion of one molecule attacks the Cys2431 sulphur atom of the Cys2431-Cys2453 disulphide bond of another VWF molecule. Trimers and higher order oligomers can form when the Cys2451 thiolate anion of one of the VWF molecules in the dimer attacks the Cys2431-Cys2353 disulphide bond of another VWF molecule. These observations provide the basis for exploring defects in lateral VWF self-association in patients with unexplained haemorrhage or thrombosis.