Identification of a Binding Site for Integrin αIIbβ3 in the von Willebrand factor (VWF) A1 Domain: Dual Roles for the A1 Domain in Platelet Thrombus Formation.

In 1999, Wu et al found that blood from patients with type 3 von Willebrand disease (lacking VWF in both plasma and platelets) could not form thrombi on a collagen surface (Arterioscler. Thromb. Vasc Biol2000, 201661–1667). This suggested that VWF was absolutely required for the accumulation of platelets in thrombi under flow, even in the presence of fibrinogen. Platelets have two VWF receptors, the GP Ib-IX-V complexes and αIIbβ3 , the former mediating the initial tethering and attachment of platelets onto VWF and the latter being involved in platelet-platelet contacts. GP Ib-IX-V binds VWF within the A1 domain and αIIbβ3 is known to bind an Arg-Gly-Asp (RGD) sequence in the C1 domain. In the study of Wu et al, reconstitution of the VWF-deficient plasma with recombinant VWF missing the A1 domain failed to restore thrombus formation, even when the collagen surface was first coated with wild-type VWF to allow platelet attachment. The A1 domain is thus important not only for initial platelet adhesion but also for thrombus accumulation, possibly by binding another platelet receptor. Consistent with this, the number of binding sites for the isolated A1 domain on the platelet surface is more than twice the number of GP Ibα polypeptides. The receptor responsible for these binding sites is unknown but αIIbβ3 is a good candidate given its high copy number and the marked defect seen in platelet thrombus formation in its absence or blockade. Of interest, while deletion of A1 prevented thrombus formation in the studies of Wu et al, mutation of the VWF RGD sequence did not. We therefore examined whether αIIbβ3 also binds within the VWF A1 domain. We found the following. 1) Purified, unactivated αIIbβ3 binds to immobilized A1 domain, binding blocked by antibodies to either αIIbβ3 or A1. 2) Unactivated αIIbβ3 does not interact with immobilized full-length VWF, but binds VWF in the presence of ristocetin. The binding of αIIbβ3 to both VWF and isolated A1 is blocked by the αIIbβ3 antibody c7E3 but not by RGD peptides, and by the A1 antibody 6G1. This suggests that the αIIbβ3 binding site in the A1 domain may overlap the 6G1 epitope (residues 700-709), which is distinct from the GPIbα binding site. 3) 6G1 inhibits shear-induced platelet aggregation—a process that requires both GP Ibα and αIIbβ3—without blocking GP Ibα binding. 4) Platelets firmly adhere on the surface containing A1 and cross-linked collagen-related peptide (CRP), a potent GP VI agonist, at high shear stresses. The CRP-GP VI interaction is not strong enough to arrest platelets under flow, suggesting that GP VI signals could activate αIIbβ3, and αIIbβ3 could mediate firm adhesion. Consistent with this, the αIIbβ3 antibody c7E3 prevented firm platelet adhesion. In summary, we find that αIIbβ3 binds to the A1 domain, in or near the sequence of Glu700-Asp709. In addition to its apparent role in platelet-platelet interactions during thrombus growth, the binding of αIIbβ3 to the VWF A1 domain may also facilitate the binding of GP Ibα to a distinct region of A1, as the site of αIIbβ3 overlaps the binding site of ristocetin and 6G1, both which induce VWF to bind GP Ibα. Therefore, by binding to the same site as 6G1 and ristocetin in the C-terminal peptide of A1, αIIbβ3 may regulate the affinity of A1 for GP Ibα in flowing blood.