Subject(s)
Factor IX/genetics , Hemophilia B/genetics , Sequence Deletion , Adult , Base Sequence , Genes , Humans , Male , Molecular Sequence DataABSTRACT
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 molecular weight than normal factor IX. A mutation G-->A at position 6365 of the gene was demonstrated by DNA sequencing and confirmed by restriction mapping which showed absence of a Hae III site. This leads to the substitution of glutamine for arginine at position -4 of the propeptide. Factor IX Strasbourg 2 was purified from plasma by DEAE Sepharose chromatography and immunoaffinity and relative to normal factor IX, binding of calcium to the mutant protein was clearly reduced in calcium lactate agarose gel. Quantification of gamma-carboxyglutamic acid residues gave about 50% carboxylation as compared to normal factor IX. Microsequencing of the NH2-terminal part of factor IX Strasbourg 2 confirmed the attachment of the propeptide and the mutation Arg-->Gln. Activation 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, factor IX Strasbourg 2 circulates with the attached propeptide and shows reduced gamma-carboxylation and delayed activation by factor XIa but a normal capacity to activate factor X after total cleavage by factor XIa.
Subject(s)
Factor IX/genetics , Factor IX/metabolism , Factor XIa/metabolism , Point Mutation , 1-Carboxyglutamic Acid/analysis , Adult , Amino Acid Sequence , Base Sequence , Blood Protein Electrophoresis , DNA Mutational Analysis , DNA Primers/genetics , Factor IX/isolation & purification , Female , Hemophilia B/blood , Hemophilia B/genetics , Humans , In Vitro Techniques , Male , Molecular Sequence Data , PedigreeABSTRACT
Protein S is the vitamin K-dependent cofactor of activated protein C which functions as a potent anticoagulant by degrading activated factors V and VIII in a Ca2+ and phospholipid-dependent reaction. Protein S circulates under two forms, free (approximately 40%) or bound to C4b-binding protein (C4b-bp); only the free form supports the cofactor activity for activated protein C. Total protein S antigen is usually measured by rocket immunoelectrophoresis. Free protein S antigen is measured by the same technique but after precipitation of the protein S-C4b-bp complex by PEG 8000. However, these immunological assays do not detect functional alterations of protein S which can be responsible for thrombosis. This paper describes a functional assay for free protein S based on its ability to promote the prolongation of clotting time following factor Va inactivation by activated protein C when coagulation is triggered by factor Xa. Using this assay a prolongation of about 100 s between 0 and 1 U/ml protein S is measured, allowing a reliable and rapid determination of functional protein S. The correlation coefficient between functional protein S and free antigenic protein S is 0.921. This functional protein S assay has allowed the detection of 34 cases of protein S deficiency, confirmed by immunological assays, and their classification. The striking observation is the high frequency (approximately 25%) of arterial thrombosis in these patients. The rapid determination of functional protein S in patients with venous or arterial thrombosis is of diagnostic interest and should allow the detection of mutant protein S in combination with an immunological assay.
