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A rapid, simple, and reproducible method for the preparation of porcine thrombin was investigated.Porcine prothrombin was isolated by carrying out isoelectric precipitation. Porcine thrombin was prepared from prothrombin which was activated by the prothrombinase complex containing Factor Ⅹa, Factor Ⅴ, Ca2+ and phospholipids.Factor Ⅹa and Factor Ⅴ were isolated by DEAE-cellulose chromatography respectively. They were both identified by SDS-PAGE. Yields and specific activity of porcine thrombin were 107 μg per milliliter plasma and 4 U per milligram protein.