VON-WILLEBRAND-FACTOR WITHOUT THE A2 DOMAIN IS RESISTANT TO PROTEOLYSIS

von Willebrand factor (vWF) is a complex multimeric plasma glycoprotei n, that plays a critical role in the mediation of platelet adhesion to the damaged vascular wall, and functions as a carrier protein for fac tor VIII. wWF has a domain structure consisting of repeated A, B, C, a nd D domains. The A1 domain is involved in binding to the platelet rec eptor glycoprotein (GP) Ib, and the A3 domain has a binding site for c ollagen. A function of the A2 domain has not been described, although point mutations identified in von Willebrand disease (vWD) type 2A pat ients are localized in this domain. To study the role of the A2 domain a deletion mutant was constructed which lacked the A2 domain, Delta A 2-vWF. Previous studies have shown that this approach is a powerful to ol to study the function of a domain in a protein since it does not af fect the activity of other domains. After expression in baby hamster k idney (BHK) cells, Delta A2-vWF was compared to wild-type (WT) vWF, an d to Delta A1-vWF (Lankhof et al., Blood 86: 1035, 1995). Ristocetin i nduced platelet binding was slightly increased but botrocetin induced platelet binding was normal as was binding to heparin and collagen typ e III. Adhesion studies to surface coated purified Delta A2-vWF or to Delta A2-vWF preincubated on collagen under flow conditions showed no abnormalities. Incubation with normal human plasma showed that Delta A 2-vWF like WT-vWF was not sensitive to proteolysis. After addition of urea, WT-vWF becomes sensitive to the protease, indicating that unfold ing of the molecule is necessary for exposure of the cleavage site. De lta A2-vWF tested under the same conditions was resistant, indicating that the protease sensitive site is located in the A2 domain.