Solid-phase immunoradiometric assays specific for Factor VIII coagulant antigen (VIII:CAg) and Factor VIII related antigen (VIIIR:Ag) have been used to assay the immunoreactivity of these antigens in plasma and whole blood during coagulation at physiological calcium concentration. When non-anticoagulated plasma, prepared from blood immediately after venipuncture, was incubated at 37°C, the concentration of VIII:CAg and VIlIR:Ag did not change. However, when whole blood, collected withoutanticoagulant, was incubated, the concentration of VIII:CAg gradually decreased to 50% of the initial value whereas the concentration of VIIIR:Ag remained unchanged. Gelchromatographic analyses revealed that coagulation of plasma leads to progressive dissociation of VIII:CAg from the factor VIII:VWF complex. When plasma was chromatographed before the onset of coagulation, VIII:CAg was eluted at the void volume together with VIIIR:Ag whereas after coagulation of the plasma VIII:CAg devoid of VIIIR:Ag was eluted after the void volume. Similarly, when the supernatant plasma from blood was chromatographed before the onset of coagulation, VIII:CAg together with VIIIR:Ag was eluted at the void volume whereas during and after coagulation the amount of VIII:CAg associated with VIIIR:Ag gradually decreased. However, no concomitant increase of the concentration of dissociated VIII:CAg was noted under the latter conditions. It seems likely, therefore, that adherance of dissociated VIII:CAg to cellular constituents accounts for the loss of VIII:CAg during coagulation of blood. On the other hand, it can not be excluded that cellular enzymes, extruded during coagulation, affect the antibody-binding capacity of VIII:CAg.Further studies indicate that, at least in part, dissociation of the factor VIII:VWF complex during coagulation is mediated by thrombin.
