The interaction of factor VIII with von Willebrand factor (vWF) was investigated on a quantitative and qualitative level. Binding characteristics were determined using a solid phase binding assay and protection of factor VIII by vWF from inactivation by activated protein C (aPC) was studied using three different assays. Deletion mutants of vWF, a 31-kD N-terminal monomeric tryptic fragment of vWF that contained the factor VIII binding site (T31) and multimers of vWF of different size were compared with vWF purified from plasma. We found that deletion of the A1, A2, or A3 domain of vWF had neither an effect on the binding characteristics nor on the protective effect of vWF on factor VIII. Furthermore, no differences in binding of factor VIII were found between multimers of vWF with different size. Also, the protective effect on factor VIII of vWF was not related to the size of the multimers of vWF. A 20-fold lower binding affinity was observed for the interaction of T31 with factor VIII, and T31 did not protect factor VIII from inactivation by aPC in a fluid-phase assay. Comparable results were found for a mutant of vWF that is monomeric at the N- terminus (vWF-dPRO). The lack of multimerization at the N-terminus may explain the decreased affinity of T31 and vWF-dPRO for factor VIII. Because of this decreased affinity, only a small fraction of factor VIII was bound to T31 and to vWF-dPRO. We hypothesized that this fraction was protected from inactivation by aPC but that this protection was not observed due to the presence of an excess of unbound factor VIII in the fluid phase. Therefore, vWF, T31, and vWF-dPRO were immobilized to separate bound factor VIII from unbound factor VIII in the fluid phase. Subsequently, the protective effect of these forms of vWF on bound factor VIII was studied. In this approach, all forms of vWF were able to protect factor VIII against inactivation by aPC completely. We conclude, in contrast with earlier work, that there is no discrepancy between binding of factor VIII to vWF and protection of factor VIII by vWF from inactivation by aPC. The protective effect of T31 was not recognized in previous studies due to its low affinity for factor VIII. The absence of multimerization observed for T31 and vWF- dPRO may explain the low affinity for factor VIII. No other domains than the binding site located at the D′ domain were found to be involved in the protection of factor VIII from inactivation by aPC.

Summaryvon Willebrand factor (vWF) is a complex multimeric plasma glycoprotein, that plays a critical role in the mediation of platelet adhesion to the damaged vascular wall, and functions as a carrier protein for factor VIII. vWF has a domain structure consisting of repeated A, B, C, and D domains. The A1 domain is involved in binding to the platelet receptor glycoprotein (GP) lb, and the A3 domain has a binding site for collagen. A function of the A2 domain has not been described, although point mutations identified in von Willebrand disease (vWD) type 2A patients are localized in this domain. To study the role of the A2 domain a deletion mutant was constructed which lacked the A2 domain, ΔA2- vWF. Previous studies have shown that this approach is a powerful tool to study the function of a domain in a protein since it does not affect the activity of other domains. After expression in baby hamster kidney (BHK) cells, ΔA2-vWF was compared to wild-type (WT) vWF, and to ΔAl-vWF (Lankhof et al., Blood 86: 1035,1995). Ristocetin induced platelet binding was slightly increased but botrocetin induced platelet binding was normal as was binding to heparin and collagen type III. Adhesion studies to surface coated purified ΔA2-vWF or to ΔA2-vWF preincubated on collagen under flow conditions showed no abnormalities. Incubation with normal human plasma showed that ΔA2-vWF like WT-vWF was not sensitive to proteolysis. After addition of urea, WT-vWF becomes sensitive to the protease, indicating that unfolding of the molecule is necessary for exposure of the cleavage site. ΔA2- vWF tested under the same conditions was resistant, indicating that the protease sensitive site is located in the A2 domain.

To assess the relative importance of the glycoprotein (GP) Ib binding domain and the RGDS binding site in platelet adhesion to isolated von Willebrand factor (vWF) and to collagen preincubated with vWF, we deleted the A1 domain yielding delta A1-vWF and introduced an aspartate- to-glycine substitution in the RGDS sequence by site-directed mutagenesis (RGGS-vWF). Recombinant delta A1-vWF and RGGS-vWF, purified from transfected baby hamster kidney cells, were compared with recombinant wild-type vWF (WT-vWF) in platelet adhesion under static and flow conditions. Purified mutants were coated on glass or on a collagen type III surface and exposed to circulating blood in a perfusion system. Platelet adhesion under static condition, under flow conditions, and in vWF-dependent adhesion to collagen has an absolute requirement for GPIb-vWF interaction. The GPIIb/IIIa-vWF interaction is required for adhesion to coated vWF under flow conditions. Under static condition and vWF-dependent adhesion to collagen, platelet adhesion to RGGS-vWF is similar as to WT-vWF, but platelet spreading and aggregation are abolished.

With the use of monoclonal antibodies that inhibit the ristocetin- induced binding of von Willebrand factor (VWF) to platelets and the binding to collagen, we have previously identified two distinct tryptic fragments. To prove that these fragments contain the platelet binding or the collagen binding domain, we investigated the direct binding of tryptic fragments of 125I-VWF to platelets in the presence of ristocetin and to collagen fibrils. During the course of the tryptic digestion, there was a rapid and parallel decrease in binding to platelets and collagen. In the first ten minutes, binding decreased greater than 50%; a further decrease to 19% and 29%, respectively, was noted at 90 minutes, but no further decrease was observed thereafter. The bound fragments were eluted from platelets and collagen and analyzed on polyacrylamide gradient gels. The fragments bound to the platelets appeared to be reduced, probably by endogenous reducing substances from the platelets. This was prevented by addition of N- ethylmaleimide during the incubation. After 24 hours of digestion, platelets predominantly bound fragments of 116 kd and collagen bound a single fragment of 48 kd. These fragments are similar to those previously identified with the monoclonal antibodies.