THE ARG-4 MUTANT FACTOR-IX STRASBOURG-2 SHOWS A DELAYED ACTIVATION BYFACTOR XIA

We have characterized at the DNA and protein levels a mutant factor IX , factor IX Strasbourg 2, which is responsible for a severe form (< 0. 01 U/ml) of haemophilia B. Factor IX Strasbourg 2 has a higher molecu lar weight than normal factor IX. A mutation G --> A at position 6365 of the gene was demonstrated by DNA sequencing and confirmed by restri ction mapping which showed absence of a Hae III site. This leads to th e substitution of glutamine for arginine at position -4 of the propept ide. Factor IX Strasbourg 2 was purified from plasma by DEAE Sepharose chromatography and immunoaffinity and relative to normal factor IX, b inding of calcium to the mutant protein was clearly reduced in calcium lactate agarose gel. Quantification of gamma-carboxyglutamic acid res idues gave about 50% carboxylation as compared to normal factor IX. Mi crosequencing of the NH2-terminal part of factor IX Strasbourg 2 confi rmed the attachment of the propeptide and the mutation Arg --> Gln. Ac tivation of factor IX Strasbourg 2 by purified factor XIa was found to be retarded as compared to normal factor IX, but after activation the mutant factor IXa was able to activate factor X. In conclusion, facto r IX Strasbourg 2 circulates with the attached propeptide and shows re duced gamma-carboxylation and delayed activation by factor XIa but a n ormal capacity to activate factor X after total cleavage by factor XIa .