Preliminary results on the carbohydrate moiety of factor VIII/von Willebrand factor (FVIII/vWf)

Abstract

Human FVIII/vWf, purified 9 000 fold, was prepared from therapeutic concentrates by gel filtration and by immuno-affinity chromatography on insolubilized immunoglobulins isolated from a rabbit immunized with the plasma of a patient devoid of FVIII R:Ag. These preparations which contain coagulant activity and agglutinate normal washed human platelets in the presence of ristocetin are immunologically pure. The carbohydrate moiety of this highly purified FVIII/vWf was submitted to analysis by gas liquid chromatography and thin layer chromatography before and after hydrazinolysis and alkaline-borohydride treatment. The total carbohydrate content is 14.4 p. cent (w/w). Man and GalNAc residues were identified, this result indicating the coexistence of N- and O-glycosidically linked glycans (70 and 30 p. cent respectively). After hydrazinolysis it was demonstrated that the N-glycosidically linked glycans do not contain GalNAc residues. One major glycan belonging to the N-acetyllactosaminic type with a bi-antennary structure has been characterized by thin layer chromatography. The alkaline-borohydride treatment procedure reduced all the FVIII/vWf GalNAc to GalNAc-ol residues, demonstrating that they are all involved in the linkage of the O-glycans with the peptide chain and consequently they cannot be in oligosaccharidic sequences inducing A-blood group activity. Furthermore, at least 10 O-glycosidically linked glycans were identified by thin layer chromatography. Thus, the high degree of heterogeneity of the FVIII/vWf carbohydrate moiety requires further structural studies in order to precise which class of glycans is involved in the biological activity of FVIII/vWf.