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Research has been conducted on metal ions Ca (super)2+ and Mn (super)2+ which are important for cofactor activity generation following factor VIII reconstitution from its isolated light and heavy chains. Results demonstrate that similar to Ca (super)2+, Mn (super)2+ has a high-affinity binding site for factor VIII which does not affect the affinity of heavy or light chains of factor VIII heterodimer.

Research has been conducted on the factor VIII interactions with Ca (super)2+. Results indicate the existence of Ca (super)2+ binding sites in factor VIII and that Ca (super)2+ must be bound to either heavy or light chain to generate active factor VIII.

Research has been conducted on the metal ion-dependent heterodimer factor VIII, a plasma protein participating in the blood coagulation cascade. The differential effect of the metal ions on the factor VIII subunit association and cofactor activity generation has been investigated.

An acidic-rich segment in the A1 domain of factor VIII that functions in the coordination of Ca(Super 2+), an ion necessary for cofactor activity is identified. Mutagenesis studies reveal that replacement of residue Glu113 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay.

We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca²+, an ion necessary for cofactor activity [Wakabayashi et al. (2004) J. Biol. Chem. 279, 12677-12684]. Mutagenesis studies showed that replacement of residue Glul 13 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay. Mutagenesis at this site suggested that substitution with relatively small, nonpolar residues was well tolerated, whereas replacement with a number of polar or charged residues appeared detrimental to activity. Ala substitution resulted in the greatest enhancement, yielding an ∼2-fold increased specific activity. Time course experiments following reaction with thrombin revealed similar rates of activation and inactivation of E1 13A as observed for the wild type. Results from factor Xa generation assays showed minimal differences in kinetic parameters and factor IXa affinity for E1 13A and wild-type factor VIIIa when run in the presence of synthetic phospholipid vesicles, whereas factor VIIIa E1 13A displayed an ∼4-fold greater affinity for factor IXa compared with factor VIIIa wild type in reactions run on the platelet membrane surface. This latter effect may be attributed, in part, to a 2-fold increased affinity of factor VIIIa E1 13A for the platelet membrane. Considering that low levels of factors VIIIa and IXa are generated during clotting in plasma, the increased cofactor specific activity observed for E1 13A factor VIII may result from its enhanced affinity for factor IXa on the physiological membrane. [ABSTRACT FROM AUTHOR]

Metal ions, such as Ca[sup 2+] and Mn[sup 2+], 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 Mn[sup 2+] showed saturable binding with high affinity (K[sup d] = 5.7 ± 2.1 µM) as detected using a factor Xa generation assay. No significant competition between Ca[sup 2+] and Mn[sup 2+] for factor VIII binding (K[sub i] = 4.6 mM) was observed as measured by equilibrium dialysis using 20 µM Ca[sup 2+] and 8 µM factor VIII in the presence of 0-1 mM Mn[sup 2+]. 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 Mn[sup 2+] (K[sub d] = 53.0 ± 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K[sub d] = 53.8 ± 14.2 nM). The sensitization of phosphorescence of Tb[sup 3+] bound to factor VIII subunits was utilized to detect Mn[sup 2+] binding to the subunits. Mn[sup 2+] inhibited the phosphorescence of Tb[sup 3+] 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[sub i] values = 169-1147 µM), whereas Ca[sup 2+] showed no effect on Tb[sup 3+] phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn[sup 2+] binding to site(s) on factor VIII that are different from the high-affinity Ca[sup 2+] binding site. However, like Ca[sup 2+], Mn[sup 2+] did not alter the affinity for HC and LC association. Thus, Mn[sup 2+] appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca[sup 2+] following its association at nonidentical sites on the protein. [ABSTRACT FROM AUTHOR]

Analyzes the binding of calcium with heavy and light chains of Factor VIII plasma protein. Mechanism of calcium-dependent conformational change; Dissociation of heavy and light protein chains; Importance of calcium in cofactor activity.