Structure, Biology, and Genetics of Factor VIII. Factor VIII Function, страница 4

The interaction of factor VIII with vWF is mediated by a major factor VIII-binding site that resides within the first 272 amino acids of the mature vWF molecule. MLID87250449 MLID88025605 MLID89292189  58–60 Specific missense mutations within this region of vWF can cause autosomal hemophilia due to a deficiency in factor VIII. MLID92336160  49 Most vWF molecules contain one factor VIII-binding site that can be saturated in vitro. MLID88087045  61 However, the ratio of factor VIII to vWF observed in vivo is 1:50. MLID77207496  44

The corresponding vWF binding site on factor VIII resides within the NH2 terminus of the light chain of factor VIII. MLID87246689 MLID88273151  62–65 A vWF-binding site on factor VIII was localized by monoclonal antibody inhibition to residues 1,673 to 1,684. MLID88186813 MLID89193471  66,67 This region is composed of a high density of acidic amino acids located at the NH2 terminus of the factor VIII light chain and is removed by thrombin cleavage at residue 1,689 (Fig. 105-1). Deletion of the acidic region in the NH2 terminus of the light chain by site-directed mutagenesis yielded a molecule that did not bind vWF with high affinity, although the purified protein had a specific activity similar to wild-type factor VIII. MLID92011498  57 These results demonstrate that the acidic region within residues 1,649–1,689 of the light chain is critical for appropriate interaction with vWF but is not required for cofactor function of factor VIII. It was recently demonstrated that antibodies that bind the factor VIII C2 domain can inhibit binding to vWF and to phosphatidylserine. MLID93227170  68 This observation is consistent with the presence of a phospholipid-binding region between residues 2,303 and 2,332 within the C2 domain MLID90248570  69 and that phospholipid and vWF compete for binding to factor VIII. MLID82134732  55,56 This finding indicates that although a primary vWF-binding site resides between residues 1,648 and 1,689, multiple contacts are probably required to mediate the multitude of effects that vWF has on factor VIII.

Factor VIII is post-translationally modified by sulfation on tyrosine residues 346,718,719,721, 1,664, and 1,680. MLID92207952  25 Inhibition of tyrosine sulfation by treatment of factor VIII-expressing cells with sodium chlorate did not affect factor VIII secretion but reduced the specific activity of the factor VIII by fivefold, indicating that this modification is required for full cofactor activity. MLID92207952  25 The importance of this post-translational modification was also studied by the conservative mutation of tyrosine residues to phenylalanine residues in order to block sulfation. Tyrosine to phenylalanine mutations at residues 346 and 1,664 reduced the rate of thrombin cleavage and activation. 70 Tyrosine to phenylalanine mutation at residue 1,680 reduced interaction with vWF by fivefold. MLID91093266  70,71 In addition, a patient with the 1,680 tyrosine-to-phenylalanine mutation had a fivefold reduction in factor VIII antigen and activity, likely due to a defect in vWF binding. MLID90152691  72 These experiments show that post-translational sulfation of tyrosine residues affects factor VIII procoagulant activity and interaction with vWF.

Binding to Other Coagulation Factors

Due to the ability of thrombin-activated human factor VIIIa, most binding studies have been performed with intact factor VIII. Since it is known that factor VIII activation influences the activity of factor IXa, MLID92381008  8 it must be considered that changes occur in binding affinities or sites of interactions (or in both) on factor VIII activation. Monoclonal antibody inhibition experiments suggest that a factor IXa-binding site exists between residues 1,770 and 1,840 of the factor VIII light chain. MLID94171722  73 In addition, factor IXa inactivates factor VIII by cleavage at residue 336 in the heavy chain MLID93104509  74,75 and prevents dissociation of the A2-domain polypeptide from thrombin-activated factor VIII. MLID92156105  76 Factor IXa protects factor VIII from inactivation by activated protein C, MLID87126793 MLID85072180  77–79 indicating possible common sites of interaction. In sum, these results suggest that factor IXa interacts with both the heavy and light chains of factor VIII. Similar characteristics were identified for the binding of enzyme factor Xa to factor Va where both the heavy chain MLID85054836 MLID87242916  80,81 and light chain MLID83108837 MLID83169885  82,83 contribute to binding. There is suggestive evidence that factor X interacts with factor VIII. MLID80130523 MLID88087235  84,85 However, no studies have localized the site of binding. Factor Va mediates binding of the substrate prothrombin via the factor V heavy chain. MLID85054836  80