Using the method of Weinstein et al. (Proc.Natl.Acad.Sci. USA# 78, 5137-41, 1981) the FVIII polypeptide distribution in wet and dry heated concentrates, a monoclonal-purified concentrate and fresh plasma was examined. Samples were incubated for 2 hours at 37°C (in the presence of polyethyleneglycol 4000 to aid complex formation) with 125I-Fab' fragments prepared from a polyclonal human anti-FVIII :C antibody. The complexes were elec-trophoresed in a 3-9% polyacrylamide gradient gel, in the presence of SDS, under non-reducing conditions and visualised by autoradiography.Fresh plasma slowed a range of peptide bands of apparent M.Wt. 80-280 kD, wicn a major band at 280 kD. FVIII concentrates showed a similar range of bands and, for one manufacturer's product (product E), an additional strong band of 40-50 kD. The proportion of total EVIII antigen in the 280 kD band was estimated by densitometry to be 20-40% in concentrates, can-pared with 65% in fresh plasma. Severe haemophilic plasma had no bands, confirming the specificity of the technique. FVIII antigen in 'wet' heated concentrates was shown to be more degraded (increase in low molecular weight forms) than in dry heated concentrates.Fresh plasma incubated at 37°C for 24 hours shewed increased amounts of FVIII antigen in a low molecular weight form (90 kD).Treatment of concentrates and plasma with thrombin resulted in a change of the peptide band pattern, which was dependent upon thrombin concentration and incubation time. Loss of the 280 kD band and intensification of a 90 kD band was observed, which correlated with an increase in FVIII :C by one-stage assay. Further proteolysis resulted in a band of inactive material of 40-50 kD, with identical mobility to the band seen in product E. FVIII :C activity in product E was higher by one-stage than by two-stage assay, and these results suggest more extensive thrombin degradation in this product.The results show that the molecular form of FVIII in concentrates is dependent upon storage of plasma, the methodof concentrate preparation and the type of heat treatment.
