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

Abstract

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ib␣ and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIb␣ binding and recognizes an epitope in the amino acids 764 -1035 region in the N-terminal DD3 domains. In this study we demonstrated that the DD3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the DD3 region in VWF augmented binding to GPIb␣, suggesting an inhibitory role for this region, (ii) the isolated DD3 region inhibited the GPIb␣ interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the DD3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIb␣. Our results support a model of domain translocation in VWF that allows interaction with GPIb␣. The suggested shielding interaction of the A1 domain by the DD3 region then becomes disrupted by VWF immobilization. The plasma protein von Willebrand factor (VWF)4 has a central role in normal primary hemostasis (1). The interaction of VWF with its platelet receptor glycoprotein (GP) Ib␣ in the GPIb-IX-V complex mediates platelet adhesion to extracellular matrices exposed at sites of vascular injury. This interaction is essential for thrombus formation at sites of high shear stress, as in microarterioles or in stenosed arteries.Mature VWF comprises a series of multimers that are composed of homodimers interlinked through disulfide bridges. The mature VWF subunit consists of four distinct types of internal homology present in two to three copies in the following order from the N terminus:

O-linked glycosylation of von Willebrand factor modulates the interaction with platelet receptor glycoprotein Ib under static and shear stress conditions

“…The DЈA3 fragment was chosen because it does not form multimers; therefore, it can be used for kinetic studies in which spatial separation of the A1 domains is essential, while still encompassing the OLGs and structural elements that have been suggested to influence accessibility of the A1 domain. 31,32 We first confirmed that all the DЈA3 mutants bound to the flow slides to a similar extent as wtDЈA3. Immobilized proteins were recovered from the slides and analyzed by SDS-PAGE followed by Western blotting and immunostaining with both polyclonal anti-VWF antibody ( Figure 2C) and anti-c-myc tag antibodies ( Figure 2D).…”

N-terminal Flanking Region of A1 Domain in von Willebrand Factor Stabilizes Structure of A1A2A3 Complex and Modulates Platelet Activation under Shear Stress

“…The primary structure of the mature vWF protein describes the A1 domain as being flanked by the DЈD3 domains in the N-terminal region, and those domains have been described to be involved in the mechanism that inhibits the vWF-GPIb␣ interaction by shielding the A1 domain (22). However, the binding site for GPIb␣ in the A1 domain remained cryptic in our A1A2A3 protein, which excludes the DЈD3 domains (4).…”

Platelet glycoprotein Ibα forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF

“…These contacts may allow the D3 and/or other domains to regulate the forced dissociation of A1 from GPIbα in a manner similar to that of the salt bridges between D1269 and R1306/R1450. This may explain why deleting the D′D3 region augments binding to GPIbα and adding an isolated D′D3 region inhibits GPIbα binding to vWF lacking this region (48). Deleting the D′D3 region (residues 764-1259) may affect the D1269:R1306/R1450 salt bridges, causing a gain-of-function phenotype like that of R1450E A1.…”