Purification of the factor VIII complex

Abstract

Two high purity factor VIII concentrates, type I and type II were developed for clinical trials in patients with hemophilia A and von Willebrand's disease. Fresh frozen plasma containing 1% polyethylene glycol 4000 was thawed to form cryoprecipitate, which was subsequently dissolved in citrate buffer. By addition of glycine buffer to a final concentration of 2.0 M at 26 degrees C, the bulk of fibrinogen was precipitated while factor VIII remained in solution. Factor VIII was precipitated from the glycine supernatant by addition of solid sodium chloride. The recovery of factor VIII procoagulant activity (VIII:C) per kg plasma was 271 +/- 23 units (n = 4) and 386 +/- 47 units (n = 7) for the type I and the type II preparations, respectively, while the recovery of von Willebrand factor related activity (ristocetin cofactor, VIIIR:RC) was 518 +/- 75 units and 718 +/- 90 units per kg plasma, respectively. The specific activity (units per mg protein) of VIII:C in the type I and type II preparations were 2.53 +/- 1.02 and 7.56 +/- 1.33, respectively. The specific activity (units per mg protein) of VIIIR:RC for the type I and type II preparations were 4.86 +/- 2.32 and 13.6 +/- 3.7, respectively. VIIIR:Ag was present as multimers in both preparations, and the multimeric pattern was similar to that of normal plasma. The preparations have the ability to correct the prolonged bleeding time in severe von Willebrand's disease. The factor VIII complex in the type II preparations was further purified by gel filtration on Sephacryl S-1000. This preparation was free of fibrinogen and fibronectin. Its specific activity in terms of VIII:C was 47 u/mg protein and 104 u/mg protein in terms of VIIIR:RC. The subunit of reduced factor VIIIR:Ag had Mr of 210 Kd on sodium dodecyl sulfate polyacrylamide gel electrophoresis.