Platelet-bound ristocetin aggregation factor in normal subjects and patients with von Willebrand's disease

Antiserum specific for that part of the factor VIII complex designated ristocetin aggregation factor (VIII_RAF) was prepared by immunizing rabbits with VIII_RAF derived from the plasma of a hemophilic patient with circulating antibody to factor VIII procoagulant activity (VIII_coag). The rabbit antiserum prevented ristocetin-induced platelet aggregation and platelet retention by glass-bead columns. Although the antiserum also inactivated VIII_coag in normal plasma, it did not inactivate VIII_coag which had been dissociated from VIII_RAF by chromatography in 1 M NaCl, thus establishing the antigenic specificity of these two factors. When the VIII_RAF antibody was conjugated with fluorescein isothiocyanate and used to examine platelets from normal subjects and patients with von Willebrand's disease, the normal platelets showed a granular fluorescence similar to that observed with antifibrinogen serum while von Willebrand platelets showed no fluorescent staining. The normal platelets retained the VIII_RAF granules during 18 hours incubation and 5 washings in artificial medium while the von Willebrand platelets failed to acquire granules after 18 hours in normal plasma. When the unstained platelets from patients with von Willebrand's disease were suspended in normal plasma and then aggregated by the addition of ristocetin, the aggregates not only stained brightly for VIII_RAF, but fluorescent granules could be seen on individual platelets in the aggregates. These observations suggest that ristocetin causes the binding of VIII_RAF to platelets as well as platelet-to-platelet adhesion.