Molecular characterization of commercial porcine factor VIII concentrate.

Commercial porcine factor VIII concentrate (Hyate:C) is effective in treatment of patients with hemophilia A who have circulating antibodies to factor VIII. The molecular forms of factor VIII in the concentrate were identified and evaluated in light of the known properties of porcine and human factor VIII. The factor VIII in the concentrate was isolated by tandem chromatography on gelatin-Sepharose and monoclonal anti-factor VIII-Sepharose. The factor VIII was 1% of the protein mass of the concentrate when calculated by either quantity of protein recovered or by radioimmunoassay. Both functional assay and Western blotting of the crude concentrate indicated that maximum coagulant function was achieved by proteolytic activation of procofactor forms of factor VIII. The factor VIII can be fractionated by cation-exchange high-performance liquid chromatography (HPLC) into two or three species of heterodimers depending on the lot. The specific activity of the purified porcine factor VIII was 550 U/mg using pooled porcine plasma at 1 U/mL as a standard. From this value, a factor VIII concentration in normal pig plasma of 2 micrograms/mL was calculated. This agreed well with a value of 3 micrograms/mL obtained by radioimmunoassay (RIA) of factor VIII in porcine plasma. In contrast, reported values for human factor VIII average 5800 U/mg, resulting in a calculated concentration in plasma of 0.2 microgram/mL. The finding that porcine plasma contains a significantly higher circulating mass of factor VIII than human plasma appears to explain previous difficulties in comparing porcine and human factor VIII in standard assays.

[1]  C. Fulcher,et al.  Characterization of the human factor VIII procoagulant protein with a heterologous precipitating antibody. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[2]  E. Chen,et al.  Characterization of the human factor VIII gene , 1984, Nature.

[3]  A. Mackay,et al.  The effect of prestaining prior to immunoreaction during electrophoretic blotting of proteins , 1987 .

[4]  H. Stormorken,et al.  [Treatment of hemophiliacs with antibodies to factor VIII]. , 1986, Tidsskrift for den Norske laegeforening : tidsskrift for praktisk medicin, ny raekke.

[5]  J. Katzmann,et al.  Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein. , 1982, Blood.

[6]  P. Lollar,et al.  Activation of porcine factor VIII:C by thrombin and factor Xa. , 1985, Biochemistry.

[7]  G. Knutson,et al.  Molecular cloning of a cDNA encoding human antihaemophilic factor , 1984, Nature.

[8]  J. Morrissey,et al.  Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. , 1981, Analytical biochemistry.

[9]  P. Kajenski,et al.  Degradation of coagulation proteins by an enzyme from Malayan pit viper (Akistrodon rhodostoma) venom. , 1987, Biochemistry.

[10]  E. Davie,et al.  Preparation and properties of bovine factor VIII (antihemophilic factor). , 1980, Biochemistry.

[11]  V. Marder,et al.  The size of human factor VIII heterodimers and the effects produced by thrombin. , 1986, Biochimica et biophysica acta.

[12]  E. Bidwell.  The Purification of Antihaemophilic Globulin from Animal Plasma , 1955, British journal of haematology.

[13]  A. Goodall,et al.  Purification of human factor VIII:C and its characterization by Western blotting using monoclonal antibodies. , 1985, Biochemistry.

[14]  U. K. Laemmli,et al.  Cleavage of structural proteins during , 1970 .

[15]  L. Hoyer.  The factor VIII complex: structure and function. , 1981, Progress in clinical and biological research.

[16]  P. Mannucci,et al.  Use of Porcine Factor VIII in the Management of Seventeen Patients with Factor VIII Antibodies , 1984, Thrombosis and Haemostasis.

[17]  T. Griggs,et al.  Purified human factor VIII procoagulant protein: comparative hemostatic response after infusions into hemophilic and von Willebrand disease dogs. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Jzn,et al.  Determination of absorption coefficients of purified proteins by conventional ultraviolet spectrophotometry and chromatography combined with multiwavelength detection. , 1985, Analytical biochemistry.

[19]  B. Keyt,et al.  Structure of human factor VIII. Nature , 1984 .

[20]  B. Keyt,et al.  Expression of active human factor VIII from recombinant DNA clones , 1984, Nature.