Chromatographic purification of human proteins having anticoagulant and anti-inflammatory activity

A method for producing a protein exhibiting anticoagulant activity, comprising:

• a. homogenizing highly vascularized tissue in the presence of a pH 5 to pH 9 buffer to obtain an aqueous homogenate;
• b. removing tissue fragment from said homogenate to produce an aqueous extract;
• c. adding ammonium sulfate to the extract to approximately 20% to 50% of saturation to form a first precipitate and supernatant;
• d. adding ammonium sulfate to the supernatant to at least approximately 60% of saturation to form a second precipitate;
• e. isolating the second precipitate and dissolving said second precipitate in a suitable buffer to form a solution;
• f. reducing the salt concentration of said solution such that said reduced solution can be separated by anion-exchange chromatography;
• g. applying the solution obtained from step (f) to an anion-exchange chromatography column and collecting the fraction obtained which is not absorbed onto said column, to produce a non-absorbed fraction,
• h. fractionating said non-adsorbed fraction by gel filtration to produce an enriched fraction having anticoagulant activity;
• i. reducing the salt concentration of said enriched fraction such that said reduced fraction can be fractionated by cation-exchange chromatography;
• and
• j. fractionating said reduced fraction by cation-exchange chromatography to separate the protein having anticoagulant activity from the reduced fraction.