Interaction of factor VIII clotting protein with phospholipids
It was shown that addition of procoagulant phospholipid (PL) vesicles to F.VIII concentrate protected F.VIII:C from anti-F.VIII:C antibody inactivation, and that of the naturally-occurring PLs only phosphatidylserine (PS)-containing PL had this ability. By using a PL-F.VIII mixture as immunoadsorbent for anti-F.VIII:Ag 125I-Fab' label, an immunoradiometric assay (IRMA) for F.VIII:Ag was set up which showed sensitivity to the binding of the antigen to PL vesicles: studies showed that only PS and phosphatidic acid (PA) vesicles could bind F.VIII:Ag, and ultracentrifugation of F.VIII-PL mixtures confirmed their physical association. The PL-fractionation method for IRMA label preparation was further refined, supplying two different pools of anti-F.VIII:Ag antibody: one (PL-site) directed only against PL-binding sites on the antigen, the other against other epitopes of F.VIII:Ag. Competition studies with the non-PL-site label showed that both purified F.IXa and F.X interacted with F.VIII:Ag in the presence of procoagulant PL and Ca2+ ions. Studies using the PL-site label (a specific assay for F.VIII-PL binding) confirmed that only the negatively-charged PS and PA were highly active in binding F.VIII:Ag, zwitterionic PLs being inactive: also, the Kd of the F.VIII-PS interaction decreased sharply with increasing proportion of PS in the vesicle. Further investigations using free-flow electrophoresis demonstrated a strong correlation between the Kd for F.VIII-PL and the net (negative) surface charge of the vesicles. However it was also shown that neither negative charge nor PS head-group alone were sufficient to bind F.VIII:Ag: both had to be present. Assay of the PL-site antibody for anti-F.VIII:C activity confirmed that the PL-binding site on F.VIII:Ag plays an important part in the protein's procoagulant function: both SDS-PAGE analysis of F.VIII:Ag/PL-site label complexes and IRM assays of purified F.VIII peptides with the two antibody pools indicated that the PL-binding site is on the 80kD light chain of the factor VIII molecule.