The Interaction of Platelet GPIB with the VWF-A1 Domain in Solution or Immobilized Is Regulated by the D3 Domain (A.A. 1238–1260) and the A2A3 Domains

We have used recombinant von Willebrand factor (VWF) fragments to investigate the influence of the flanking regions of the A1 domain on the interaction with platelet glycoprotein (GP)Ibα. One fragment, the r1261–1874, represented mainly the A1A2A3 domains and the other, r1238–1874, included the C-terminal region of the D3 domain. When immobilized onto a surface, the two fragments tethered platelets similarly at high shear rates. The two fragments bound identically to immobilized fixed platelets in a ristocetin-dependent and saturable manner. In the absence of ristocetin, the r1261–1874 had a 50% of its GPIb-binding activity with the modulator while the r1238–1874 only had < 5% binding activity. The r1261–1874 fragment effectively inhibited both ristocetin-induced platelet agglutination and shear-induced platelet aggregation. In contrast, the r1238–1874 failed to show an inhibitory capacity in the two assays, indicating a limited interaction with platelet GPIbα in solution. We also analyzed the single bond lifetimes in a range of constant forces of GPIbα with either the single A1 domain (a.a. 1238–1471) or the A1A2A3 fragment using atomic force microscopy (AFM). When the protein was directly adsorbed on a Petri dish, the GPIbα bound with longer lifetimes to the A1A2A3 than the isolated A1 domain, suggesting that a conformational change in the A1 is influenced by the A2A3 domains. In conclusion, the C-terminal region of D3 domain (a.a.1238–1260) and the A2A3 domains negatively regulate the binding of the A1 domain to platelet GPIbα in suspension. On the other hand, the A2A3 domains may have a positive influence on the bond formed between the GPIbα and a surface-bound A1 domain.