Shielding of the A1 domain by the D'D3 domains of von Willebrand factor modulates its interaction with platelet glycoprotein Ib-IX-V.
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.
Ulrichts
Hans
H
Laboratory for Thrombosis Research, Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, B-8500 Kortrijk, Belgium.
Udvardy
Miklós
M
Lenting
Peter J
PJ
Pareyn
Inge
I
Vandeputte
Nele
N
Vanhoorelbeke
Karen
K
Deckmyn
Hans
H
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*
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