Summary
A novel CRM+ mutation, factor VIII position 373 serine to leucine substitution (FVIII
373-Leu) was identified during a survey of Factor VIII (FVIII) mutations. We have
purified the variant protein from the patient's plasma in order to allow further characterisation
of the molecule. The CRM+ plasma contained 120% Factor VIII antigen (FVIILAg) and
6% Factor Vili coagulant activity (FVIILC). After purification the mutant FVIII was
subjected to thrombin proteolysis, and was thereby activated 5.6-fold compared with
7-fold for wild type molecule. Subsequently, spontaneous inactivation of the mutant
was much slower than noted for wild type FVIII. Western blot analysis using monoclonal
antibodies demonstrated that thrombin cleavage of FVIII 373-Leu at positions 740 and
1689 were normal but that cleavage at position 372 was completely absent.
Crystallographic coordinates of the active site of thrombin complexed to fibrinopeptide
A were used to explore possible mechanistic reasons for the failure of thrombin to
cleave the mutant FVIII at position 372. Steric hindrance between the mutant side
chain and the side chain of the PI residue was apparent. We conclude that the functional
defect of FVIII 373-Leu results from the inability of thrombin to cleave the mutant
at position 372-373, and propose that this is due to steric hindrance by the side
chain of leucine 373, preventing correct formation of the enzyme substrate complex.
