The deduced amino acid sequence of human factor VIII, obtained from the DNA sequence, predicts a mature polypeptide of 2,332 amino acids containing a triplicated domain structure. The polypeptide has 35% sequence homology with the copper-binding plasma protein, ceruloplasmin. Determination of the thrombin cleavage sites in plasma-derived factor VIII polypeptides allows prediction of the domains involved in the associated activation and inactivation of the protein.

Factor VIII was purified from cryoprecipitate by ion exchange chromatography on solid phase polyelectrolyte E-5 (PE-E5). The product was highly purified (3.5 u VIII:C/mg protein) compared to conventional concentrate (0.3 u VIII:C/mg protein) with low fibrinogen, low isoagglutinin titre, and a ratio of factor VIII coagulant activity (VIII:C) to factor VII related antigen (VIIIR:Ag) of 16:1. Trial infusions of this material (PE VIII) were given to three patients with severe haemophilia A and one patient with homozygous von Willebrand's disease. These patients also each received separate infusions of intermediate purity concentrate (IPC) for comparison. There were no adverse effects. The mean half life of VIII:C after PE VIII infusion in the haemophiliacs was 10.9 h and after IPC was 12.1 h, a statistically insignificant difference. The survival of factor VIII coagulant antigen (VIII:CAg) was similar to that of VIII:C. In contrast, the half life of VIII:C and of VIII:CAg was very short after infusion of PE VIII in the patient wih von Willebrand's disease (2.4 h). IPC when infused in this patient produced a typical secondary rise of VIII:C. Two bleeding episodes in severe haemophiliacs were satisfactorily treated with PE VIII. PE-E5 deserves further study as a means of preparing clinical concentrates of factor VIII.

Human factor VIII:C has been purified over 300 000-fold from cryoprecipitate by polyelectrolyte purification followed by affinity chromatography on Sepharose linked to antibody to factor VIIIR:Ag (monoclonal or polyclonal) and Sepharose linked to monoclonal antibody to factor VIII:C. The purified material has been analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies. PAGE shows predominant bands at 360K (unreduced), 210K, and 90K and an 80K/79K doublet; Western blotting showed all the monoclonal antibodies used bound the 360K form. In a small-scale purification, plasma from blood taken directly into thrombin inhibitor Kabi S-2581 was applied directly to the monoclonal anti-factor VIII:C column. Western blot analysis of this material showed the 360K band on reduction. The purified factor VIII:C could be activated 13-fold by human thrombin. Gel analysis of the activated material showed intensification followed by fading of the band at 90K and generation of bands at 70K/69K, 55K, and 40K. Western blotting shows that the 70K/69K doublet derives from the 80K/79K moiety and the 40K peptide derives from the 90K and is presumed to contain the active site. From these studies an epitope map of the factor VIII:C molecule has been constructed.

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