Factor VIII Is A Calcium-Binding Protein

The high molecular weight complex of Factor VIII is known to dissociate into two subunits when chromatographed in a high ionic strength solution containing either calcium or sodium salts. Calcium also has an essential role in maintaining Factor VIII activity during storage (AABB 1980). These two findings suggest ah integral involvement of calcium in the basic structure-function relationship of the Factor VIII molecule. Consequently, we have examined the binding of 45Ca to both the low molecular weight (LMW) procoagulant subunit of Factor VIII and the high molecular weight (HMW) complex using PAGE and column chromatography on Sepharose 4B. When the HMW complex of Factor VIII was isolated from cryoprecipitate by standard chromatographic procedures, incubated with 45CaCl2 and subjected to polyacrylamide gel electrophoresis, the resultant autoradiogram demonstrated that all of the 45Ca was associated with the HMW material which did not enter the gel. When this HMW Factor VIII was incubated with 45Ca and then dissociated by column chromatography in a buffer containing 0.25 M CaCl2, all of the 45Ca was associated with the material eluting in the 2.3 Vo region. This corresponds to the elution volume of the LMW, procoagulant subunit of Factor VIII. Dialysis against buffer was not effective in removing the 45Ca. The data indicate that calcium is tightly associated with the low molecular weight subunit of Factor VIII and presents further corroborative evidence that calcium has an essential role in determining both the chemical and physical properties of the Factor VIII molecule.