The Functional Heterogeneity Of Clinical FVIII Concentrates

There is an increasing tendency to use more highly purified FVIII preparations, and more sophisticated fractionation methods. In some cases new populations of FVIII:RAg have been produced that have a greater or lesser physiological activity. The characterisation of clinical concentrates on the basis of their biological activity is therefore extremely important in the search for more effective therapy.

We have investigated the activity of several high purity concentrates (Hemofil, Koate, Actif-VIII) as well as low purity pooled cryoprecipitate, with and without added ¹²⁵I- anti-VIII:C(*IgG or *FAB), after electrophoresis in 1% agarose gels.

In concentrates with a high FVIII:RAg concentration(25-30Iu/ml), 2 or more FVIII:RAg peaks were seen, with partial or complete identity. The main one migrated in the α 2 region, but a more anodal peak was frequently seen. The relative concentration of fast moving VIII:RAg in cryoprecipitate was too low to allow detection by immunoelectrophoresis. In the presence of *IgG or *FAB, only one main peak of radiolabelled VIII:C was noted, which corresponded with the larger molecular forms of VIII:RAg. In contrast, bioassays based on FXa generation (VIII:C and VIII:CAm), revealed 2 main peaks of activity in concentrates, cryoprecipitate and plasma. The slower moving peak correlated well with the radiolabelled VIII:C pattern. The fast moving peak in concentrate corresponded with the trailing edge of the fast moving VIII:RAg peak. This activity was not neutralised by increasing the *IgG level, or addition of anti-IXc IgG to the sample. No enzymic activity was detected in this region using a wide range of chromogenic substrates. However, Al(OH)₃ adsorption did remove the activity. These forms of FVIII may represent a native subpopulation or proteolytic breakdown product, which has lost some antigenic determinants, but retained biological activity. This material, which has a lower affinity for anti VIII:C and RAg, could be present in plasma and be selectively concentrated during fractionation procedures.