Studies on the structure and subunit composition of human antihaemophilic factor

Human antihaemophilic factor was purified by Hydroxylapatite chromatography following precipitation from plasma and gel filtration on Sepharose 6B. Application to Hydroxylapatite was in 0.01 M tris HC1 (pH 7.35) - 0.14 M NaCl and after washing with 5m M phosphate (pH 6.8) - 0.1 M NaCl the antihaemophilic factor was eluted with 0.1 M phosphate (pH 6.8) - 0.1 M NaCl. Factor VIII coagulant activity, factor VIII related antigen and von Willebrand factor activity eluted simultaneously. The protein(s) had a molecular weight in excess of 500,000 and multiple subunits as shown by electrophoresis in 5% acrylamide gels containing sodium dodecyl sulphate and 4–27% gradient acrylamide gels: without reduction the protein failed to enter these gels but following reduction multiple bands were observed, the major band had a molecular weight around 200,000. Thin layer peptide mapping demonstrated structural inter-relationship between the 200,000 dalton protein and three of the smaller species, however, other unrelated smaller species were evident. It is apparent from these findings that human factor VIII may exist as multiple molecular forms due to heterogeneity of one subunit (MW around 200,000) and the molecular structure may include other smaller non-identical subunits. The structure-function relationships of these subunits remains to be elucidated.