ATP-dependent dissociation of non-disulfide-linked aggregates of coagulation factor VIII is a rate-limiting step for secretion

Deficiency in coagulation factor VIII leads to the bleeding disorder hemoph ilia A. Previous studies demonstrated that factor VIII secretion is limited due to an ATP-requiring step early in the secretory pathway. In this repor t, we identified that this ATP-dependent rate-limiting step involves the di ssociation of non-disulfide-linked aggregates within the endoplasmic reticu lum (ER). In contrast to the numerous examples of interchain disulfide-link ed aggregates, factor VIII is the first protein characterized to form non-d isulfide-linked high molecular weight aggregates within the ER. Approximate ly a third of newly synthesized factor VIII was detected in high molecular weight aggregates. These aggregates disappeared over time as functional fac tor VIII appeared in the medium. The aggregated complexes did not require p roteasomal degradation for clearance. Aggregate formation was enhanced by A TP depletion, and upon restoration of metabolic energy, these aggregates we re dissociated and secreted. With the coexpression of von Willebrand factor (vWF), a small portion of vWF coaggregated with factor VIII. However, vWF dissociated from the aggregates more rapidly than factor VIII, supporting t hat these aggregates are dynamic. An increase in the factor VIII expression level elicited a corresponding increase in the fraction of factor VIII tha t was aggregated. In addition, a 110 amino acid sequence containing a hydro phobic beta-sheet within factor VIII was identified that may predispose fac tor VIII to aggregation. These data show that formation and ATP-dependent d issolution of nondisulfide-linked factor VIII aggregates is a dynamic, rate -limiting step during the folding process in the early secretory pathway. I n summary, we have identified an unprecedented requirement for protein tran sport out of the ER that involves an ATP-dependent dissociation of non-disu lfide-linked aggregates within the ER.