Molecular forms of antihaemophilic globulin in plasma, cryoprecipitate and after thrombin activation.

Abstract

Summary. Sedimentation characteristics of factor VIII were investigated by ultracentrifugation in 10–40% w/v sucrose gradient in pH 7.2 imidazole buffered saline at 65 000 rpm for 6 hr. Following dialysis of fractions for 12 hr against buffered saline, the factor‐VIII activity was assayed by a one‐stage method and other proteins were identified by immunological techniques. In human citrated plasma, two regions of factor‐VIII activity were detected, a main fast sedimenting activity in the region ofγM macroglobulin and a minor slow sedimenting activity in the region of fibrinogen. Cryoprecipitate activity was all of the fast type, while the activity in its supernatant was of the slow type. When plasma was fractionated in a sucrose gradient prepared in 0.4 or 1 M NaCl, the factor‐VIII activity was found in the slow fraction, suggesting that fast‐sedimenting factor VIII is a non‐covalently bound polymer. Incubation of plasma with thrombin (0.035 u/ml for 10 min at 37°C) markedly increased the factor‐VIII activity, all of which appeared in the slow fraction. Although this suggests the possibility that the fast fraction represents a factor VIII‐fibrinogen complex, immunological analysis of this fraction failed to reveal the presence of any fibrinogen. Alternatively, the fast fraction may be composed of sub‐units of factor VIII which are both released and activated by thrombin.