POSTTRANSLATIONAL REQUIREMENTS FOR FUNCTIONAL FACTOR-V AND FACTOR-VIII SECRETION IN MAMMALIAN-CELLS

Coagulation factors V and VIII are homologous glycosylated plasma prot eins that provide essential functions for hemostasis. Factor V is secr eted as a single chain polypeptide, whereas factor VIII is processed i ntracellularly to yield a metal-ion-associated heterodimer that is sta bilized through interaction with von Willebrand factor. In transfected mammalian cells, factor V is more efficiently secreted than factor VI II. To provide insight into the different secretion efficiencies, we c ompared the post-translational processing requirements for factor V an d factor VIII expressed in mammalian cells. In contrast to factor VIII , factor V was not detected in association with the immunoglobulin-bin ding protein (BiP), a chaperonin protein of the endoplasmic reticulum (ER). Depletion of intracellular ATP levels by treatment of cells with low concentrations of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) , protonophore that uncouples oxidative phosphorylation, inhibited sec retion of factor VIII but had no effect on the secretion of factor V. Inhibition of N-linked oligosaccharide addition by treatment with tuni camycin prevented secretion of both factor V and factor VIII, whereas treatment with an inhibitor of complex oligosaccharide addition, deoxy mannojirimycin, did not affect secretion, although the specific activi ties of both factor V and factor VIII were slightly increased. Thus, c omplex oligosaccharide addition was not required for secretion or func tional activity of either factor V or factor VIII. Depletion of intral umenal calcium with the ionophore A23187 did not affect secretion of e ither factor V or factor VIII. In the presence of A23187, the secreted factor V was fully functional, whereas the factor VIII heavy and ligh t chains were not associated and the secreted molecule was inactive. I n addition, A23187 treatment inhibited addition of serine/threonine (O )-linked oligosaccharides to factor V and factor VIII. The differences between factor V and factor VIII were further evaluated by characteri zation of a single chain mutant factor VIII. The single chain factor V III was secreted with an efficiency similar to wild-type factor VIII a nd also required von Willebrand factor for stabilization. In addition, the activity of single chain factor VIII was also inhibited by A23187 treatment, suggesting a unique metal-ion requirement within the secre tory pathway for functional factor VIII folding. The differences ident ified in BiP association, ATP requirements, and metal-ion dependence f or effective functional secretion of these two molecules may underlie mechanisms accounting for their different secretion efficiencies.