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Thromb Haemost 1996; 75(03): 450-455
DOI: 10.1055/s-0038-1650295
Original Article
Schattauer GmbH Stuttgart

Isolation and Characterization of Canine Factor IX

Yuichi Sugahara
1   The Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA
,
James Catalfamo
2   Comparative Hematology Section, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
,
Marjory Brooks
2   Comparative Hematology Section, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
,
Eri Hitomi
1   The Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA
,
S Paul Bajaj
3   The Departments of Medicine, Biochemistry, and Pathology, St. Louis University School of Medicine, St. Louis, MO, USA
,
Kotoku Kurachi
1   The Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA
› Author Affiliations
Further Information

Publication History

Received 07 September 1995

Accepted after revision 22 November 1995

Publication Date:
26 July 2018 (online)

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Summary

Canine plasma factor IX was purified to homogeneity by a combination of barium citrate precipitation and three-step column chromatographies of DEAE sepharose, heparin agarose and a monoclonal antifactor IX antibody-linked agarose. Canine factor IX has an apparent molecular size of 61 kDa, which is slightly smaller than that of human factor IX, as determined by denatured polyacrylamide gel electrophoresis. Its amino acid composition, amino-terminal and carboxyterminal amino acid sequences agreed well with those predicted from the reported cDNA. Unlike purified human factor IX, canine factor IX preparation often showed a discrete smaller molecular species (∼50 kDa) which was generated by a specific proteolytic cleavage between Arg310 and Val311. When purified canine factor IX was utilized as a standard for enzyme linked immunosorbent assay, the concentration of canine factor IX in the pooled normal dog plasma was determined to be 5.3 Μg/ml with 11.2% carbohydrate content (or 4.7 Μg/ml for its polypeptide chain moiety). Concentration of plasma factor IX antigen was measured in six severely affected, unrelated hemophilia B dogs. Four had factor IX antigen of less than 1% of the normal, and two had undetectable levels. The latter two had gross molecular abnormalities in their factor IX genes. Three obligate carrier females had variable but proportionately reduced factor IX antigen and factor IX coagulant activity levels. These results provide a quantitative method for measuring canine factor IX antigen which is a prerequisite for studying hemostasis and development of gene transfer approaches in the canine model of hemophilia B.

 

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