Interaction of von Willebrand Factor Domain A1 with Platelet Glycoprotein Ib alpha (1-289). Slow intrinsic binding kinetics mediate rapid platelet adhesion

We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ib alpha . Recombinant VWF A1 domain (residues Glu super(497)-Pro super(705) of VWF) bound stoichiometrically to a GPIb alpha calmodulin fusion protein (residues His super(1)-Val super(289) of GPIb alpha ; GPIb alpha CaM) immobilized on W-7-agarose with a K sub(d) of 3.3 mu M. The variant VWF A1(R545A) bound to GPIb alpha CaM 20-fold more tightly, mainly because the association rate constant k sub(on) increased from 1,100 to 8,800 M super(-1) s super(-1). The GPIb alpha mutations G233V and M239V cause platelet-type pseudo-von Willebrand disease, and VWF A1 bound to GPIb alpha (G233V)-CaM and GPIb alpha (M239V)-CaM with a K sub(d) of 1.0 and 0.63 mu M, respectively. The increased affinity of VWF A1 for GPIb alpha (M239V)-CaM was explained by an increase in k sub(on) to 4,500 M super(-1) s super(-1). GPIb approximately aCaM bound with similar affinity to recombinant VWF A1, to multimeric plasma VWF, and to a fragment of dispase-digested plasma VWF (residues Leu super(480)/Val super(481)-Gly super(718)). VWF A1 and A1(R545A) bound to platelets with affinities and rate constants similar to those for binding to GPIb alpha CaM, and botrocetin had the expected positively cooperative effect on the binding of VWF A1 to GPIb alpha CaM. Therefore, allosteric regulation by botrocetin of VWF A1 binding to GPIb alpha , and the increased binding affinity caused by mutations in VWF or GPIb alpha , are reproduced by isolated structural domains. The substantial increase in k sub(on) caused by mutations in either A1 or GPIb alpha suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The exceptionally slow k sub(on) and k sub(off) provide important new constraints on models for rapid platelet tethering at high wall shear rates.