Cleavage of factor VIII heavy chain is required for the functional interaction of A2 subunit with factor IXa

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC), Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by similar to 100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors Ma and X resulted in an increase in factor Ma activity because of conversion of the I-IC to Al and A2 subunits by factor Xa, HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of similar to1% observed for thrombin, HC showed Little inhibition of the A2 subunit-dependent stimulation of factor Ma activity, suggesting that factor Ma-interactive sites are mashed in the A2 domain of HC, Furthermore, RC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor Ma in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor Ma-interactive site(s) in the A2 subunit required to modulate protease activity.