Factor VIIIa consists of subunits designated A1, A2, and A3-C1-C2. The limited cofactor activity observed with the isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1 336 ) was previously shown to possess similar affinity for A2 and retain ϳ60% of its A2 stimulatory activity. We now identify a second site in A1 at Lys 36 that is cleaved by factor Xa. A1 truncated at both cleavage sites (A1 37-336 ) showed little if any affinity for A2 (K d >2 M), whereas factor VIIIa reconstituted with A2 plus A1 37-336 /A3-C1-C2 dimer demonstrated significant cofactor activity (ϳ30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtained by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with A1 37-336 showed little activity in a one-stage clotting assay. This resulted in part from a 5-fold increase in K m for factor X when A1 was cleaved at Arg 336 . These findings suggest that both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K m for factor X binding to factor Xase, and this parameter is critical for activity assessed in plasmabased assays.Factor VIII, a plasma protein that participates in the blood coagulation cascade, is deficient or defective in individuals with hemophilia A. Factor VIII functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa. Factor VIII is synthesized as a multi-domain, single chain molecule (A1-A2-B-A3-C1-C2) (1) with a molecular mass of ϳ300 kDa (2, 3). Factor VIII is processed to a series of divalent metal ion-linked heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1-A2-B domains and a light chain consisting of the A3-C1-C2 domains. This procofactor is activated by cleavage at Arg 372 , Arg 740 , and Arg 1689 by thrombin and factor Xa, converting the dimer into the factor VIIIa trimer composed of the A1, A2, and A3-C1-C2 subunits (4, 5). The resulting factor VIIIa heterotrimer retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits, whereas A2 is associated with a weak affinity by electrostatic interactions (5, 6). Factor VIIIa is unstable, and loss of activity is due to the dissociation of the A2 subunit from the A1/A3-C1-C2 dimer (5-7). Under physiological conditions, the K d for this interaction is ϳ260 nM (8, 9); however, at slightly acidic pH and low ionic strength, this interaction is facilitated by an ϳ10-fold increase in the affinity (K d ϭ ϳ30 nM) (8).The role of factor VIIIa in the intrinsic factor Xase is to bind factor IXa, which increases the k cat for factor Xa formation by several orders of magnitude compared with factor IXa alone (10). Interactive sites for factor IXa are localized to A2 and A3 domains (11-13). Recent studies have shown that modulation of factor IXa by the isolated A2...

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Previously, we demonstrated that Ca(2+) was necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC) but that Ca(2+) did not affect HC-LC binding affinity (Wakabayashi et al. (2001) Biochemistry 40, 10293-10300). Titration of EDTA-treated factor VIII with Ca(2+) followed by factor Xa generation assay showed a two-site binding pattern, with indicated high-affinity (K(d) = 8.9 +/- 1.8 microM) and low-affinity (K(d) = 4.0 +/- 0.6 mM) sites. Analysis by equilibrium dialysis using (45)Ca and <400 microM free Ca(2+) verified a high-affinity binding (K(d) = 18.9 +/- 3.7 microM). Preincubation of either HC or LC with 6 mM Ca(2+) followed by reassociation with the untreated complementary chain in the presence of 0.12 mM Ca(2+) failed to generate significant cofactor activity (<0.5 nM min(-1) (nM LC)(-1)). However, pretreatment of both HC and LC with 6 mM Ca(2+) followed by reassociation (at 0.12 mM Ca(2+)) generated high activity (7.5 +/- 0.4 nM min(-1) (nM LC)(-1)). Progress curves for activity regain following factor VIII-Ca(2+) association kinetics fitted well to a series reaction scheme rather than one of simple association (p < 0.0001), suggesting a multistep process which may include a Ca(2+)-dependent conformational change. These results suggest that factor VIII contains two Ca(2+) binding sites with different affinities and that active factor VIII can be reconstituted from HC and LC only when both chains are preactivated by Ca(2+).

Factor VIIIa consists of three subunits designated A1, A2, and A3-C1-C2. The isolated A2 subunit possesses limited cofactor activity in stimulating factor IXa-catalyzed activation of factor X. This activity is markedly enhanced by the A1 subunit (inter-subunit K d ‫؍‬ 1.8 M). The C-terminal region of A1 subunit (residues 337-372) is thought to represent an A2-interactive site. This region appears critical to factor VIIIa, because proteolysis at Arg 336 by activated protein C or factor IXa is inactivating. A truncated A1 (A1 336 ) showed similar affinity for A2 subunit (K d ‫؍‬ 0.9 M) and stimulated its cofactor activity to ϳ50% that observed for native A1. However, A1 336 was unable to reconstitute factor VIIIa activity in the presence of A2 and A3-C1-C2 subunits. Fluorescence anisotropy of fluorescein (Fl)-FFR-factor IXa was differentially altered by factor VIIIa trimers containing either A1 or A1336 . Fluorescence energy transfer demonstrated that, although Fl-A1 336 /A3-C1-C2 bound acrylodan-A2 with similar affinity as the native dimer, an increased inter-fluorophore separation was observed. These results indicate that the C-terminal region of A1 appears necessary to properly orient A2 subunit relative to factor IXa in the cofactor rather than directly stimulate A2 and elucidate the mechanism for cofactor inactivation following cleavage at this site.Factor VIII is a protein cofactor for the serine protease factor IXa, which catalyzes the conversion of factor X to Xa. Factor VIII is synthesized as a multidomainal single-chained molecule (A1-A2-B-A3-C1-C2) (1), with a molecular mass of ϳ300 kDa (2, 3). Factor VIII circulates as a partial proteolyzed protein containing a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains), which are held together by a metal iondependent linkage. This procofactor is activated by thrombin, converting the dimer into a trimer composed of the A1, A2, and A3-C1-C2 subunits (4, 5). The resulting factor VIIIa heterotrimer retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits whereas a weak affinity electrostatic interaction exists between A1 and A2 (5, 6). Factor VIIIa is considerably unstable, and loss of activity is due to the dissociation of the A2 subunit from the A1/A3-C1-C2 dimer (5-7).Under physiological conditions, the K d for this interaction is ϳ260 nM (8, 9), however, under slightly acidic, pH and lower ionic strength, A2 and A1/A3C1C2 can be reconstituted to form the trimer with a K d of ϳ30 nM (8).The role of factor VIIIa is to bind factor IXa, generating the phospholipid-dependent intrinsic factor Xase complex, which increases the k cat for factor Xa formation by several orders of magnitude compared with factor IXa alone (10). At least two interactive sites have been identified for the enzyme-cofactor interaction. A high affinity, surface proximal site is likely formed by residues contained in the A3 domain and light chain of factors VIIIa and IXa, respectively, whereas a weaker affinity interaction involves residues localized to the A...

Metal ions, such as Ca2+ and Mn2+, are necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC). Titration of EDTA-treated factor VIII with Mn2+ showed saturable binding with high affinity (K(d) = 5.7 +/- 2.1 microM) as detected using a factor Xa generation assay. No significant competition between Ca2+ and Mn2+ for factor VIII binding (K(i) = 4.6 mM) was observed as measured by equilibrium dialysis using 20 microM Ca2+ and 8 microM factor VIII in the presence of 0-1 mM Mn2+. The intersubunit affinity measured by fluorescence energy transfer of an acrylodan-labeled LC (fluorescence donor) and fluorescein-labeled HC (fluorescence acceptor) in the presence of 20 mM Mn2+ (K(d) = 53.0 +/- 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K(d) = 53.8 +/- 14.2 nM). The sensitization of phosphorescence of Tb3+ bound to factor VIII subunits was utilized to detect Mn2+ binding to the subunits. Mn2+ inhibited the phosphorescence of Tb3+ bound to HC and LC, as well as the HC-derived A1 and A2 subunits with a relatively wide range of estimated inhibition constant values (K(i) values = 169-1147 microM), whereas Ca2+ showed no effect on Tb3+ phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn2+ binding to site(s) on factor VIII that are different from the high-affinity Ca2+ binding site. However, like Ca2+, Mn2+ did not alter the affinity for HC and LC association. Thus, Mn2+appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca2+following its association at nonidentical sites on the protein.