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The C2 domain of factor VIII (FVIII) is important for FVIII–phospholipid (PL) and FVIII–von Willebrand factor (VWF) interactions. A FVIII structural model, derived by electron crystallography, suggests four hydrophobic loops at the FVIII C2 domain–PL interface. Within loop four, the solvent-exposed amino acid, Trp2313, is believed to contribute to FVIII–PL binding. To analyse this interaction, the amino-acid exchange Trp2313 to Ala (W2313A) was introduced into the C2 domain of B-domain-deleted FVIII (dBFVIII). Both proteins, dBFVIII and W2313A, were expressed in a mammalian expression system. Labelling experiments showed that the mutation W2313A resulted in reduced secretion but did not affect intracellular synthesis of the protein. Specific activity, kinetic parameters, binding to VWF and haemostatic potential in a murine model of haemophilia A were found to be similar for both proteins. Binding studies to synthetic 4% phosphatidyl- l-serine vesicles showed, however, a 28-fold higher K D for W2313A, indicating the important role of Trp2313 in the FVIII–PL interaction. In conclusion, the C2-domain-surface-exposed residue Trp2313, is critical for secretion of the protein. The W2313A mutation weakens binding to phosphatidyl- l-serine vesicles but the mutant protein has the same effector function as dBFVIII in vitro and in vivo. [ABSTRACT FROM AUTHOR]

We constructed factor VIII-heparin cofactor II (FVIII-HCII) hybrid molecules, which are more readily activated by thrombin in vitro than the respective wild-type molecules. The hybrid proteins were tested in a murine model of haemophilia A to investigate their haemostatic efficacy in vivo. Bleeding characteristics, measured using standard tail-tip cutting techniques, were total blood loss, bleeding time and survival rate. FVIII-HCII hybrids were found to be effective in preventing bleeding in FVIII knockout mice. While in vitro experiments showed that the chimaeric molecules had higher haemostatic functions than the wild-type proteins, the variables analysed in vivo were similar for both proteins. [ABSTRACT FROM AUTHOR]