Metal ion-independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter-subunit affinity.

Factor VIII circulates as a divalent metal ion-dependent heterodimer comprised of a light chain (LC) and a heavy chain (HC). Reassociation of factor VIII subunits was assessed using fluorescence energy transfer where LC and HC were labeled with acrylodan (Ac; fluorescence donor) and fluorescein-5-maleimide (Fl; fluorescence acceptor), respectively. The reduction of donor fluorescence due to the acceptor was used as an indicator of binding. Subunits associated with high affinity (K(d) = 53.8 nM) in the absence of metal ion and presence of EDTA. However, this product showed no cofactor activity, as measured by a factor Xa generation assay. In the presence of 25 mM Ca(2+), no increase in the intersubunit affinity was observed (K(d) = 48.7 nM) but specific activity of the cofactor was approximately 30% that of native factor VIII. At saturating levels of Fl-HC relative to Ac-LC, donor fluorescence decreased to 79.3 and 73.5% of its original value in the absence and presence of Ca(2+), respectively. Thrombin cleaved the heterodimers that were associated in the absence or presence of Ca(2+) with similar efficiency, indicating that the lack of activity was not the result of a defect in activation. Cu(2+) (0.5 microM) increased the intersubunit affinity by approximately 100 fold (K(d) = 0.52 nM) and the specific activity to approximately 60% of native factor VIII. The former effect was independent of Ca(2+), whereas the latter effect required Ca(2+). These results indicate that the intersubunit association in factor VIII is primarily metal-ion independent while divalent metal ions serve specific roles. Ca(2+) appears essential to promote the active conformation of factor VIII while Cu(2+) primarily enhances the intersubunit affinity.

Factor VIIIa cofactor activity shows enhanced ionic strength sensitivity in the absence of phospholipid.

Factor VIIIa, a cofactor for the protease factor IXa, is a trimer of A1, A2 and A3-C1-C2 subunits. In the absence of phospholipid (PL), the k(cat) for factor VIIIa-dependent, factor IXa-catalyzed conversion of factor X was markedly less than that observed in the presence of PL (approx. 150 min(-1)) and decreased as the ionic strength of the reaction increased. At low salt concentration, the k(cat) (5.5 min(-1)) was approx. 8-fold greater than observed at near physiologic ionic strength (0.7 min(-1)). However, this level of salt showed minimal effects on the intermolecular affinities of factor VIIIa (or isolated A2 subunit) for factor IXa or on the K(m) for factor X. Alternatively, the association of A2 subunit with A1 subunit was sensitive to increases in salt and paralleled the reduction in k(cat) observed with factor VIIIa. This instability was not observed in PL-containing reactions. Fluorescence energy transfer between acrylodan-A2 and fluorescein-A1/A3-C1-C2 dimer showed a requirement for both PL and factor IXa for maximal association of A2 with dimer. These results indicate that in the presence of factor IXa, the salt-dependent dissociation of factor VIIIa subunits is significantly enhanced in the absence of PL, promoting a reduced k(cat) for the cofactor-dependent generation of factor Xa.

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 approximately 100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of approximately 1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa 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 IXa-interactive site(s) in the A2 subunit required to modulate protease activity.

Sites in the A2 subunit involved in the interfactor VIIIa interaction.

Factor VIIIa is a trimer of the A1, A2, and A3-C1-C2 subunits. Regions in the A2 subunit that interact with the A1/A3-C1-C2 dimer were localized using synthetic peptides derived from A2 sequences showing high probability of being surface exposed. Peptides were restricted to residues 373-562 of A2 based on the earlier observation that this region of A2 reacts with A1 using a zero length cross-linker. Peptides were assessed for their capacity to inhibit the reconstitution of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit. Reconstitution was monitored using both regeneration of factor VIIIa activity and fluorescence quenching of an acrylodan-labeled A2 (Ac-A2) by fluorescein-labeled A1/A3-C1-C2. The activity assay identified four peptides as inhibitors, residues 373-395 (IC(50) = 65 micrometer), 418-428 (IC(50) = 25 micrometer), 482-493 (IC(50) = 325 micrometer), and 518-533 (IC(50) = 585 micrometer). The 373-395 and 518-533 peptides eliminated the fluorescence quenching of Ac-A2, whereas the 418-428 peptide reduced but did not eliminate Ac-A2 quenching. Peptide 482-493 had no effect on the fluorescence quenching of Ac-A2 suggesting that the peptide did not directly affect reassociation of the factor VIIIa subunits. These results identify three regions in the A2 subunit (373-395, 418-428, and 518-533) that interact with the A1/A3-C1-C2 dimer. Furthermore, comparison of results obtained using the two assays distinguish inhibition of the intersubunit interactions from intermolecular interactions.