Human Thrombins

Human o-thrombin, the thromboplastin activation product of prothrombin with high clotting and e&erase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 + 1.3 mg/ml with a yield of 340 2 110 mg/kg of paste, which represented 48 + 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 f 0.4 U.S. (NIH) unitslpg of protein and titrated to 88 + 8% active with p-nitrophenyl-p’-guanidinobenzoate (NPGB). Those (N = 29) examined by labeling with [Wldiisopropyl phosphorofluoridate (iPr,P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (IV = 4) or predominantly cY-thrombin (97 * 3%) and corresponding amounts of its degradation product, /?-thrombin (2.6 + 3.1%). No plasmimogen), prothrombin complex factors (II, VII, IX, IX,, X, X,), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCI, pH 6, thrombin was stable for -’ 1 week at 4” and for >l year at s-50”; freeze-dried thrombin stored at 4” for >l year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [‘CliPr,P-F, >95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH,-terminal residues were released in three consecutive Edman degradation cycles.