title: 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) 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.
authors.affiliation: Laboratory for Thrombosis Research, Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, B-8500 Kortrijk, Belgium.
authors.forename: Hans
authors.initials: H
authors.lastname: Ulrichts
authors.forename: Miklós
authors.initials: M
authors.lastname: Udvardy
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Lenting
authors.forename: Inge
authors.initials: I
authors.lastname: Pareyn
authors.forename: Nele
authors.initials: N
authors.lastname: Vandeputte
authors.forename: Karen
authors.initials: K
authors.lastname: Vanhoorelbeke
authors.forename: Hans
authors.initials: H
authors.lastname: Deckmyn
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001792:Blood Platelets / Q000378:metabolism; D024061:Collagen Type III / Q000737:chemistry; D006224:Cricetinae; D003435:Crotalid Venoms / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012310:Ristocetin / Q000737:chemistry; D013552:Swine; D013997:Time Factors; D014841:von Willebrand Factor / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D024061:Collagen Type III; D003435:Crotalid Venoms; D017931:DNA Primers; D000939:Epitopes; D019038:Platelet Glycoprotein GPIb-IX Complex; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D012313:RNA; C030961:botrocetin