The von Willebrand factor self-association is modulated by a multiple domain interaction.
BACKGROUND
Platelet adhesion and aggregation at sites of vascular injury exposed to rapid blood flow require von Willebrand factor (VWF). VWF becomes immobilized by binding to subendothelial components or by a self-association at the interface of soluble and surface-bound VWF.


OBJECTIVE
As this self-association has been demonstrated only under shear conditions, our first goal was to determine whether the same interaction could be observed under static conditions. Furthermore, we wanted to identify VWF domain(s) important for this self-association.


RESULTS
Biotinylated VWF (b-VWF) interacted dose-dependently and specifically with immobilized VWF in an enzyme-linked immunosorbent assay (ELISA) assay, showing that shear is not necessary to induce the VWF self-association. Whereas anti-VWF monoclonal antibodies (mAbs) had no effect on the self-association, the proteolytic VWF-fragments SpII(1366-2050) and SpIII(1-1365) inhibited the b-VWF-VWF interaction by 70 and 80%, respectively. Moreover, a specific binding of b-VWF to immobilized Sp-fragments was demonstrated. Finally, both biotinylated SpII and SpIII were able to bind specifically to both immobilized SpII and SpIII. Similar results were observed under flow conditions, which confirmed the functional relevance of our ELISA system.


CONCLUSIONS
We have developed an ELISA binding assay in which a specific VWF self-association under static conditions can be demonstrated. Our results suggest a multiple domain interaction between immobilized and soluble VWF.
Ulrichts
H
H
KU Leuven Campus Kortrijk, Kortrijk, Belgium.
Vanhoorelbeke
K
K
Girma
J P
JP
Lenting
P J
PJ
Vauterin
S
S
Deckmyn
H
H
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D019921:Biotinylation; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014841:von Willebrand Factor / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D014841:von Willebrand Factor; D006493:Heparin