REQUIREMENTS OF VON-WILLEBRAND-FACTOR TO PROTECT FACTOR-VIII FROM INACTIVATION BY ACTIVATED PROTEIN-C

The interaction of factor VIII with von Willebrand factor (VWF) was in vestigated on a quantitative and qualitative level. Binding characteri stics 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 3 1-kD N-terminal monomeric tryptic fragment of vWF that contained the f actor VIII binding site (T31) and multimers of vWF of different size w ere 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 c haracteristics nor on the protective effect of vWF on factor VIII, Fur thermore, no differences in binding of factor VIII were found between multimers of vWF with different size. Also, the protective effect on f actor 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 inactiv ation 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 l ack of multimerization at the N-terminus may explain the decreased aff inity of T31 and vWF-dPRO for factor VIII, Because of this decreased a ffinity, only a small fraction of factor VIII was bound to T31 and to vWF-dPRO, We hypothesized that this fraction was protected from inacti vation by aPC but that this protection was not observed due to the pre sence of an excess of unbound factor VIII in the fluid phase. Therefor e, vWF, T31, and vWF-dPRO were immobilized to separate bound factor VI II from unbound factor VIII in the fluid phase, Subsequently, the prot ective 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 ag ainst inactivation by aPC completely. We conclude, in contrast with ea rlier work, that there is no discrepancy between binding of factor VII I 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 multimerizatio n observed for T31 and vWF-dPRO may explain the low affinity for facto r VIII. No other domains than the binding site located at the D' domai n were found to be involved in the protection of factor VIII from inac tivation by aPC. (C) 1996 by The American Society of Hematology.