Abstract
Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha 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/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha 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 D'D3 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 GPIbalpha. Our results support a model of domain translocation in VWF that allows interaction with GPIbalpha. The suggested shielding interaction of the A1 domain by the D'D3 region then becomes disrupted by VWF immobilization.
Highlights
The plasma protein von Willebrand factor (VWF)4 has a central role in normal primary hemostasis [1]
Human VWF in solution does not interact with its platelet receptor GPIb-IX-V under normal conditions
Binding is induced by immobilization of VWF or by exposure to shear. These observations suggest that the affinity toward GPIb-IX-V is regulated by conformational changes in VWF that are induced by shear and/or immobilization and lead to exposure of functional sites
Summary
The plasma protein von Willebrand factor (VWF) has a central role in normal primary hemostasis [1]. Conformational changes in VWF upon immobilization or upon exposure to shear might relieve this possible shielding effect of neighboring domains in VWF and might thereby reveal the functional binding site in the A1 domain This mode of action is supported by kinetic and crystal studies, which demonstrated a higher binding affinity of the isolated A1 domain for GPIb-IX-V compared with full-length VWF (9 –11). The N-terminal region in the A1 domain (aa 1260 –1271), flanking the disulfide bridge, modulates the A1/GPIb␣ interaction; (i) crystal studies reveal a displacement of this flanking region upon binding to GPIb␣ [10, 11]; (ii) naturally occurring mutations in this region have been reported to induce von Willebrand disease (VWD) type 2B phenotype, characterized by an increased affinity of VWF for GPIb␣ [13]; (iii) Ala substitution mutants in this region show an increased affinity for GPIb␣ [14]
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