The von Willebrand factor (VWF) is a huge multimeric protein that plays a key role in hemostasis. It triggers platelet adhesion in areas of vascular damage by binding to exposed sub-endothelial collagen and thus causes wound closure. Sites for binding to collagen, an initial step of hemostasis, are located in domains A1 and A3 of VWF. Collagen III is believed to interact with the A3-domain, and collagen VI with the A1-domain. The forces and the dynamics of these interactions were investigated with single molecule force spectroscopy (SMFS), using AFM tips functionalized with VWF and substrates containing a dense layer of collagen. We also investigated three mutations in the A3-domain of the VWF A1-A2-A3 construct (S1731T, Q1734H and H1786R). Interactions between collagen type III or VI and the S1731T mutant showed no significant difference in stability when compared to the wild type construct. The A3-domain mutations Q1734H and H1786R formed a slightly more stable complex with collagen III, probably due to additional hydrogen bonds.
The same mutants on collagen VI resulted in a drastic increase in bond stability. Thus VWF domain A1 might compensate for mutations in domain A3 which could explain the inconspicuous bleeding tendency found in patients carrying these VWF A3 domain mutations.
VWF domain A1 is also responsible for mediating platelet adhesion under flow through the platelet glycoprotein Ib[alpha] (GPIb[alpha]). The adjacent domain A2 is unfolded under shear upon which it exposes a proteolytic site that is cleaved by the metalloprotease ADAMTS13 to prevent thrombosis. In the resting state, i.e. under low shear-stress conditions, VWF is incapable of binding platelets. This behavior has been associated with a shielding of the GPIb[alpha] binding site in domain A1 by domain A2. Nevertheless, the exact shielding mechanism has not been clarified yet. We thus probed the interaction strength between the A1 and A2 domains and the unfolding behavior of A2 using SMFS.
Domain A2 remained largely folded during the dissociation process from A1, keeping the VWF protected against cleavage and degradation.
In summary, the intermolecular binding between VWF and collagen and intramolecular VWF domain association processes were characterized to elucidate key initial steps in hemostasis.