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 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 ϳ100-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 ϳ1% 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.Factor VIII, the plasma protein deficient or defective in individuals with hemophilia A, is synthesized as a 300-kDa precursor (1, 2) with the domain structure A1-A2-B-A3-C1-C2 (3). It is processed to a series of divalent metal ion-dependent heterodimers after cleavage at the B-A3 junction, generating a HC 1 (A1-A2-B domains) and a LC (A3-C1-C2 domains). Additional cleavage sites within the B domain result in variably sized HCs minimally represented by contiguous A1-A2 domains. The two chains can be separated by chelating reagents (4, 5) and isolated after ion exchange and/or immunoaffinity chromatography. Factor VIII activity can be reconstituted from the separated chains by combining them in the presence of a divalent metal ion (6).Factor VIII functions in the intrinsic factor Xase complex as a cofactor for the serine protease, factor IXa in the surface-dependent conversion of factor X to Xa. This activity is dependent upon conversion of factor VIII to the active cofactor form, factor VIIIa, by thrombin or factor Xa. These enzymes cleave factor VIII HC at Arg-740, removing the B domain (or fragments) and at Arg-372, bisecting the HC into the A1 and the A2 subunits (7). The proteases also cleave factor VIII LC at Arg-1689 (7), liberating an acid-rich region and creating a new NH 2 terminus. Thus, factor VIIIa is a heterotrimer of subunits designated as A1, A2, and A3-C1-C2 (8, 9). The A1 and A3-C1-C2 subunits retain the divalent metal ion-dependent linkage, whereas the A2 subunit is weakly associated with the A1-A3-C1-C2 dimer by primarily electrostatic interactions (9, 10).Two regions of factor VIII have been identified as interactive sites for factor IXa. A high affinity site (K d ϳ15 nM (11)) was localized to the A3 domain of the LC in and around residues 1811-1818 (...

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The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830].