Dissociation of factor VIII procoagulant antigen VIII:CAg and factor VIII related antigen VIIIR:Ag by EDTA - influence of divalent cation on the binding of VIII:CAg and VIIIR:Ag.

Assuming 1 U/ml in citrated plasma, the VIII:CAg concentration was found 1.66 U/ml in EDTA-plasma, 1.09 U/ml in heparinized plasma and 0.67 U/ml in serum. Addition of 10 mmol/l EDTA to citrated and heparinized plasmas increased VIII:CAg 1.5fold. There was no increase of VIII:CAg in serum. Gel filtration of plasmas on different anticoagulants showed an elution of VIII:CAg in the void volume Vo and in the later fractions. The VIII:CAg amount detected in the internal volume increased following the series heparin less than citrate less than EDTA. Serum VIII:CAg was eluted at 2.2 Vo. Presence of EDTA in the elution buffer or incubation of plasma with EDTA prior to chromatography caused a displacement of practically all VIII:CAg amount in the internal volume with a peak at 2.2-2.3 Vo. VIIIR:Ag was exclusively detected in the void volume. Removal of divalent cation by chelation likely exposes more antigenic determinants of VIII:CAg, which are otherwise masked by steric hindrance due to VIIIR:Ag in citrated and heparinized milieu. Moreover gel filtration of plasma in the presence of EDTA completely dissociates VIII:CAg from VIIIR:Ag. The VIII:CAg fragment, having an estimated molecular weight of 70,000, might also be present in serum.