von Willebrand factor (VWF) serves a critical role in hemostasis by facilitating the initial tethering of platelets to the subendothelium at high shear and as a carrier protein for factor VIII . VWF is synthesized by endothelial cells and megakaryocytes and circulates in blood as a series of multimers made up of a variable number of disulphide linked 500 kDa homodimers .
The Weibel-Palade bodies in endothelial cells and the α-granules of platelets release ultra-large VWF in response to vascular injury and platelet activation. The largest VWF multimers have a molecular mass in excess of 20 000 kDa. The larger the VWF multimer the higher avidity it has for platelets and subendothelium. A selective deficiency of these forms is associated with a bleeding diathesis, type IIA von Willebrand disease .
The only known mechanism of controlling VWF multimer size is shear-dependent proteolysis at the Tyr842-Met843 peptide bond by the liver enzyme, ADAMTS-13 (adisintegrin-like and metalloprotease with thrombospondin type 1 motif) [4–6]. ADAMTS-13 hydrolyses the ultra-large VWF multimers as they emerge from endothelial cells and undergo conformational strain because of shear stress in arterioles and capillaries . ADAMTS-13 was identified from its association with thrombotic thrombocytopenic purpura (TTP).
TTP is a thrombotic microangiopathy characterized by a schistocytic hemolytic anemia and consumptive thrombocytopenia with varying degrees of neurological and renal impairment. The presence of ultra-large VWF multimers in the plasmas of patients with chronic relapsing TTP during remission that disappear during an attack led to the implication of these multimers in the pathogenesis of the platelet rich, fibrin poor thrombi that occlude arterioles and are the hallmark of this disorder . The persistence of unprocessed ultra-large VWF multimers in the circulation is thought to precipitate platelet clumping in arterioles and capillaries resulting in tissue ischemia . A severe deficiency of ADAMTS-13 is associated with congenital and acquired forms of TTP [8–10].
Despite the strong association of ADAMTS-13 activity with TTP and the presence of ultra-large VWF multimers in the plasma, there is still significant processing of VWF multimer size in ADAMTS-13 null mice  and in the absence of ADAMTS-13 in humans . Moreover, there is no current evidence that processing of multimer size in the absence of ADAMTS-13 is due to other proteolysis of the VWF. These findings indicate that there is another mechanism of control of VWF multimer size.
A possible clue to this mechanism comes from studies of α-keratin, the main protein component of hair. Two molecules of keratin form a coiled-coil dimer. Two coiled-coil dimers form a staggered tetramer and eight tetramers form a rope-like filament. The tetramers are linked by interchain disulphide bonds . The number and position of inter-tetrameric disulphide bonds dictate the structure of the keratin fibre and that of hair. Exposure of hair to a stress such as bending strains the inter-tetrameric disulphides and triggers a thiol-disulphide exchange that is catalyzed by unpaired cysteine thiols in keratin (Fig. 1A). This exchange continues until the stress across the fibre is resolved. The end result is a new arrangement of inter-tetrameric disulphides and a new hair structure [13,14].
There are some interesting similarities between α-keratin and VWF. Both are multimeric elongated proteins, the subunits of both proteins are linked by disulphide bonds and both undergo shear-dependent conformational change. Moreover, there is evidence that VWF multimer size can be influenced by thiol-mediated events. Thrombospondin-1 can change VWF multimer size by facilitating reversible reduction/oxidation of the disulphide bonds that hold VWF multimers together [15,16].
We suggest the following mechanism of control of VWF multimer size. Unprocessed VWF is released from endothelial cells or activated platelets and tethers to their surface. This sudden exposure to the high shear of flowing blood triggers a thiol-disulphide exchange of the inter-dimeric disulphides. The exchange continues until there is separation of the VWF multimer and release of the processed VWF into the blood (Fig. 1B).
For this mechanism of control of VWF multimer size to be feasible there must exist unpaired cysteine thiols in VWF. These are required to trigger thiol-disulphide exchange of the inter-dimeric disulphides (Fig. 1B). Evidence for cysteine thiols was sought by labelling purified plasma VWF with the biotin-linked maleimide, 3-(N-maleimidylpropionyl)biocytin (MPB). Maleimides specifically alkylate cysteine thiols at neutral pH. VWF incorporated MPB, indicating the presence of unpaired cysteines in the protein (Fig. 1C). These thiols were refractory to alkylation by MPB unless the protein was first denatured with sodium dodecyl sulphate (SDS) and heat, which implies that the thiols are buried to some extent in the native protein.
This hypothetical mechanism could explain the reduction in VWF multimer size observed in the absence of ADAMTS-13 in mice and humans. Attractive features of this mechanism are that the VWF processing would occur constitutively as it is released from endothelial cells and that no additional factors beyond the shear provided by flowing blood are required. It may be that this mechanism is mostly responsible for basal control of VWF multimer size, while ADAMTS-13 is important for regulating VWF multimer size in situations associated with acute platelet and/or endothelial cell activation.