Purification of low molecular weight factor VIII by affinity chromatography using factor VIII - sepharose

Abstract

Purified human FVIII coupled to Sepharose reversibly binds the procoagulant activity of soluble FVIII in a time dependent manner. The binding capacity of FVIII Sepharose prepared with 6.7 units of purified FVIII was 1.8 units of FVIII:C, of which approximately 30% could be recovered by elution with 1.3M NaCl followed by concentration with PEG. The affinity-purified FVIII corrected the clotting defect in FVIII-deficient but not in FIX-deficient plasma and retained its procoagulant activity when incubated with DFP. Its sedimentation rate in sucrose density gradients was constitent with a 7.2S protein, and it was retarded by exclusion chromatography through a column of Sepharose CL-6B, though some aggregation was apparent. No FVIII:Ag was detected by Laurell immunoelectrophoresis or by radioimmunoassay, indicating a ratio of FVIII:C/FVIII:Ag of ≥ 150. Thrombin exposure enhanced the procoagulant activity of affinity-purified FVIII, though more slowly and less extensively than that of undissociated FVIII. The thrombin enhanced activity was relatively stable.