Interaction of von Willebrand Factor Domain A1 with Platelet Glycoprotein Ibα-(1–289): SLOW INTRINSIC BINDING KINETICS MEDIATE RAPID PLATELET ADHESION

Shuji Miura,Chester Q Li,Zhaofeng Cao,Huifang Wang,Mark R Wardell,J.Evan Sadler

doi:10.1074/jbc.275.11.7539

Shuji Miura, Chester Q Li + Show 4 more

Open Access

https://doi.org/10.1074/jbc.275.11.7539

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Abstract

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

Highlights

We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ib␣
Design and Characterization of Recombinant VWF A1 and GPIb␣ Variants—Plasma VWF is multivalent, and this feature complicates the study of reactions between individual binding sites on VWF and platelet GPIb␣
Monomeric recombinant VWF A1 proteins were expressed in E. coli and refolded by a method developed previously for the renaturation of tissue-type plasminogen activator [24]

Summary

SLOW INTRINSIC BINDING KINETICS MEDIATE RAPID PLATELET ADHESION*

The substantial increase in kon caused by mutations in either A1 or GPIb␣ suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The VWD type 2B mutation I546V was shown to cause a significant change in the crystal structure of recombinant VWF domain A1, altering the positions of residues proposed to bind GPIb␣ [17]. The exceptionally slow rate of binding and the acceleration caused by mutations are consistent with a model in which rate-limiting conformational changes in both proteins are necessary for productive VWF-GPIb␣ binding. The slow rate constants for binding provide new constraints on models for how VWF mediates rapid platelet tethering under conditions of high wall shear stress in vivo

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION