Promotion of binding of von Willebrand factor to platelet glycoprotein Ib by dimers of ristocetin

Abstract

In the absence of high shear forces, the in vitro binding of human von Willebrand factor (vWF) to its platelet receptor glycoprotein Ib (GPIb) can be promoted by two well-characterized mediators, botrocetin and ristocetin. Using purified vWF and GPIb, we have investigated the mechanisms by which ristocetin mediates this binding. Specific binding of vWF monomers to GPIb occurred with a 1:1 stoichiometry, but high-affinity binding required the participation of two ristocetin dimers. Binding was strongly dependent on pH and inhibited by low poly-L-lysine concentrations, indicating ristocetin-dependent charge neutralization during the interaction. With increasing ristocetin concentrations, vWF binding depended progressively less on the involvement of its A1 loop, which is compatible with a model in which the two ristocetin dimers bridge the vWF-GPIb complex on secondary sites. In agreement with this model, the ristocetin-dimer-promoted stabilization of vWF on GPIb was abolished by low concentrations of poly(Pro-Gly-Pro), which is known to complex ristocetin dimers. Mechanistic analysis of the inhibition of vWF binding by the recombinant vWF fragment Leu504-Ser728 (VCL), which covers the entire A1 loop, revealed an affinity of VCL for GPIb comparable with that of the botrocetin-vWF complex for GPIb, and identified a specific but 20-fold lower affinity of VCL in the presence of ristocetin. The proline-rich peptides flanking the vWF A1 loop, Cys474-Val489 and Leu694-Asp709, inhibited vWF binding semispecifically by competitively interfering with the formation of the GPIb-vWF complex rather than by complexation of free ristocetin dimers. In conclusion, ristocetin-promoted binding of vWF to its GPIb receptor results from charge neutralization and interactions involving proline residues in the vicinity of the natural interaction sites present on both GPIb and the A1 domain of vWF.

Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers

“…While the exact mechanism of ristocetin-induced binding of VWF to GP Ib is not known, some have suggested that ristocetin binds to GP Ib through 4 ristocetin monomers or through 2 ristocetin dimers. 22 While 2 ristocetin dimers could indeed act as a bridge between VWF and GP Ib, it is also possible that multiple ristocetin dimers, perhaps 6, could facilitate the VWF/GP Ib interaction. Future experiments using the optical tweezers will allow us to address the nature of the ristocetin-dependent VWF/GP Ib bond.…”

“…Botrocetin forms a complex with VWF by binding near the GP Ib-binding site. 21 Ristocetin is an antibiotic glycopeptide synthesized by Nocardia lurida thought to interact with both GP Ib-IX and VWF, 22 although the mechanism by which it induces the VWF-GP Ib-IX interaction remains unclear.…”

Total inhibition of high shear stress induced platelet aggregation by homodimeric von Willebrand factor A1‐loop fragments

“…The cause of this difference is unknown. However, it is established that both agonists modulate the GPIba-binding ability differently, because botrocetin specifically binds to the A1-loop region (Sugimoto et al, 1991), but ristocetin itself dimerizes (Scott et al, 1991;Hoylaerts et al, 1995) and binds to both the flanking region of the A1-loop (Mohri et al, 1988) and GPIba (Scott et al, 1991).…”

N-Glycosylthioureido Aglyco-ristocetins without Platelet Aggregation Activity

“…The above structural elements facilitate the binding of the antibiotic to specific sequences of von Willebrand's factor (vWF) thereby inducing the interaction of vWF with platelet glycoprotein 1b (GP1b) and initiating aggregation [10]. The dimerization ability of 1 may also play an important role in this mechanism [9,11].…”