Functional modulation of the isolated glycoprotein Ib binding domain of von Willebrand factor expressed in Escherichia coli

Abstract

We have expressed in Escherichia coli the domain of von Willebrand factor (vWF) containing the binding site for platelet glycoprotein (GP) Ib and used it to study the regulation of vWF-platelet interaction. The recombinant fragment, comprising residues 445-733 of the mature vWF subunit and designated rvWF445-733, did not have the native conformation of the corresponding domain in the intact molecule because, in order to prevent formation of random aggregates, the seven cysteine residues in the sequence were reduced and alkylated. Unlike native vWF, rvWF445-733 bound to GP Ib in the absence of any modulator, suggesting that the lack of disulfide bonds and/or carbohydrate side chains within this domain may expose platelet interaction sites. In the presence of two modulators, the glycopeptide ristocetin and the snake protein botrocetin, rvWF445-733 inhibited native vWF binding to GP Ib as well as platelet aggregation mediated by vWF, suggesting that both the fragment and the native molecule interact with the same site on platelets. This conclusion was also supported by the observation that the recombinant fragment competed with the binding to platelets of an anti-GP Ib monoclonal antibody known to inhibit vWF binding. Botrocetin formed a complex with rvWF445-733, but the affinity of this interaction was approximately 25-fold lower than with native vWF. However, the complexes of botrocetin with either rvWF445-733 or multimeric native vWF bound to GP Ib with similar dissociation constant. Therefore, conformational attributes of vWF regulate its affinity for botrocetin, but once the complex is formed, interaction with GP Ib is independent of native vWF conformation. These findings provide insights into the regulation of vWF-platelet interaction.

Shielding of the A1 Domain by the D′D3 Domains of von Willebrand Factor Modulates Its Interaction with Platelet Glycoprotein Ib-IX-V

“…Dimeric constructs were used because previous studies demonstrated the necessity of the use of at least dimeric molecules for sustaining platelet aggregation (33). The threshold dose needed for ristocetin-induced agglutination of washed platelets was lower for ⌬DЈD3 than for plusDЈD3 (Fig.…”

Expression and Purification of a Soluble Recombinant A1 Domain of Human von Willebrand Factor in Bacteria

“…The A1 domain of VWF is of particular interest in view of its critical role in initiating the formation of a thrombus in a region of arterial stenosis. Several investigators have thus developed strategies to express the A1 domain of human VWF as a recombinant protein (5,6,9,12,13,15,19,20,22). Despite the large number and variety of the prior constructs, none can be judged as ideal from the perspective of yielding a recombinant A1 domain protein that retains the structural and functional features of this domain in the intact VWF macromolecule.…”

“…This is a substantive disadvantage as it necessitates denaturation of the recombinant protein with urea or guanidinium chloride to extract it from these bodies. Reduction of the disulfide bonds also proved necessary with most of those prior constructs (6,12,13,15,19,20,22). Explicit renaturation and oxidation steps were incorporated after purification in some cases (6,12,13,19,20,22).…”

“…As many difficulties arise in studying the structure and function of the A1 domain in the intact VWF molecule (10), recombinant forms of the isolated A1 domain have been generated by several laboratories. Prior to this study, at least nine distinct A1 domain constructs for bacterial expression in Escherichia coli were reported (5,6,9,12,13,15,19,20,22). The recombinant A1 domain protein, however, is usually found as a precipitate in inclusion bodies, resulting in problems with its purification.…”

The pathophysiology and management of thrombotic thrombocytopenic purpura

“…This agent is now under investigation as an arterial antithrombotic agent and may hold promise in the therapy of thrombotic microangiopathy, in which high shear stress forces in damaged microvessels may sustain vWF-mediated intravascular platelet aggregation. Recombinant fragments of the vWF monomer that competitively block the binding of vWF multimers to GPlb (143), or monoclonal antibodies to the arginine-glycine-aspartatebinding region for glycoprotein IIb/IIIa on monomeric subunits of vWF multimers (144) are also under investigation.…”