ISOLATION AND CHARACTERIZATION OF CANINE FACTOR-IX

Canine plasma factor IX was purified to homogeneity by a combination o f barium citrate precipitation and three-step column chromatographies of DEAE sepharose, heparin agarose and a monoclonal antifactor IX anti body-linked agarose. Canine factor IX has an apparent molecular size o f 61 kDa, which is slightly smaller than that of human factor IX, as d etermined by denatured polyacrylamide gel electrophoresis. Its amino a cid composition, amino-terminal and carboxyterminal amino acid sequenc es agreed well with those predicted from the reported cDNA. Unlike pur ified human factor IX, canine factor IX preparation often showed a dis crete smaller molecular species (similar to 50 kDa) which was generate d by a specific proteolytic cleavage between Arg310 and Val311. When p urified canine factor CY was utilized as a standard far enzyme linked immunosorbent assay, the concentration of canine factor IX in the pool ed normal dog plasma was determined to be 5.3 mu g/ml with 11.2% carbo hydrate content (or 4.7 mu g/ml for its polypeptide chain moiety). Con centration of plasma factor IX antigen was measured in six severely af fected, unrelated hemophilia B dogs. Four had factor IX antigen of les s than 1% of the normal, and two had undetectable levels. The latter t wo had gross molecular abnormalities in their factor IX genes. Three o bligate carrier females had variable but proportionately reduced facto r IX antigen and factor IX coagulant activity levels. These results pr ovide a quantitative method for measuring canine factor IX antigen whi ch is a prerequisite for studying hemostasis and development of gene t ransfer approaches in the canine model of hemophilia B.