PROMOTION OF FINDING OF VON-WILLEBRAND-FACTOR TO PLATELET GLYCOPROTEIN IB BY DIMERS OF RISTOCETIN

In the absence of high shear forces, the in vitro binding of human von Willebrand factor (vWF) to its platelet receptor glycoprotein Ib (GPI b) can be promoted by two well-characterized mediators, botrocetin and ristocetin. Using purified vWF and GPIb, we have investigated the mec hanism by which ristocetin mediates this binding. Specific binding of vWF monomers to GPIb occurred with a 1:1 stoichiometry, but high-affin ity binding required the participation of two ristocetin dimers. Bindi ng was strongly dependent on pH and inhibited by low poly-L-lysine con centrations, indicating ristocetin-dependent charge neutralization dur ing the interaction. With increasing ristocetin concentrations, vWF bi nding depended progressively less on the involvement of its A1 loop, w hich is compatible with a model in which the two ristocetin dimers bri dge the vWF-GPIb complex on secondary sites. In agreement with this mo del, the ristocetin-dimer-promoted stabilization of vWF on GPIb was ab olished by low concentrations of poly(Pro-Gly-Pro), which is known to complex ristocetin dimers. Mechanistic analysis of the inhibition of v WF binding by the recombinant vWF fragment Leu(504)-Ser(728) (VCL), wh ich covers the entire A1 loop, revealed an affinity of VCL for GPIb co mparable with that of the botrocetin-vWF complex for GPIb, and identif ied a specific but 20-fold lower affinity of VCL in the presence of ri stocetin. The proline-rich peptides flanking the vWF A1 loop, Cys(474) -Val(489) and Leu(694)-Asp(709), inhibited vWF binding semispecificall y by competitively interfering with the formation of the GPIb-vWF comp lex rather than by complexation of free ristocetin dimers. In conclusi on, ristocetin-promoted binding of vWF to its GPIb receptor results fr om charge neutralization and interactions involving proline residues i n the vicinity of the natural interaction sites present on both GPIb a nd the A1 domain of vWF.