A MECHANISM FOR INHIBITION OF FACTOR-VIII BINDING TO PHOSPHOLIPID BY VON-WILLEBRAND-FACTOR

von Willebrand factor (vWf) acts as a carrier for blood coagulation fa ctor VIII (fVIII) in the circulation, The amino-terminal 272 residues of mature vWf contain a high affinity fVIII binding site, Upon thrombi n activation, fVIII is released from vWf, thereby allowing its binding to phospholipid which is required for its procoagulant activity. Alth ough phospholipid and vWf compete for fVIII binding, it was previously suggested that their binding sites are not closely juxtaposed within the fVIII protein because only amino-terminal vWf proteolytic fragment s larger than SPIII-T4 (1-272) were able to block the binding of fVIII to phospholipid. We have demonstrated, however, that SPIII-T4 is able to inhibit fVIII binding to phosphatidylserine (PS) in a dose-depende nt fashion, but only at concentrations higher than those used in previ ous experiments. Our demonstration that the K-d values for vWf and SPI II-T4 for fVIII are 0.52 nM and 48 nn, respectively, explain this disc repancy. Inhibition (>95%) of SPIII-T4 binding to fVIII by a purified recombinant fVIII C2 domain polypeptide demonstrated that SPIII-T4 bin ds directly to C2, as we had previously shown for vWf, The similarity of the C2 binding sites for vWf and SPIII-T4 was further confirmed by the identical inhibitory effects of synthetic peptides and monoclonal antibodies (mAbs) on vWf-fVIII or SPIII-T4-fVIII binding, In both case s, binding was inhibited by synthetic peptide 2303-2332, containing a PS binding site, and by mAb NMC-VIII/5 Fab' (epitope within C2 residue s 2170-2327), We propose that vWf, via residues 1-272, and PS compete for fVIII binding because they recognize overlapping sites within fVII I C2 domain residues 2303-2332.