Generation of active coagulation factor VIII from isolated subunits.
Factor VIII-light chain (FVIII-LC) and Factor VIII-heavy chain (FVIII-HC) were isolated separately from human plasma and were without coagulation activity. When FVIII-LC and FVIII-HC preparations were mixed, coagulation activity was generated in the presence of Mn2+, Ca2+, or Co2+. Mn2+ was most effective and with Ca2+ maximal activity was first obtained after 8 days. Bovine Factor X (FX) accelerated recombination and was able to increase the amount of FVIII:C generated up to 10-fold when FVIII-LC/HC were incubated with Ca2+ for 20 h. When recombination was performed in the presence of micromolar concentrations of sulfhydryl reagents, the total amount of FVIII:C generated was increased up to 4-fold and in excess of FVIII-HC it was possible to drive 70% of FVIII-LC into active complex. If FVIII-HC was prepared in the presence of a sulfhydryl reagent it was possible to drive 75% of FVIII-HC into active complex with FVIII-LC in excess. Me2+, which is necessary for recombination, catalyzes disulfide formation, and it is proposed that FVIII subunits have free sulfhydryl groups. The presence of sulfhydryl reagents during purification of FVIII-HC and during recombination retains the individual subunits in a conformation suitable for recombination.
D002118:Calcium / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000096:biosynthesis*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D013997:Time Factors
D046911:Macromolecular Substances; D003035:Cobalt; D008345:Manganese; D005169:Factor VIII; D002118:Calcium