Multiple Active Forms of Thrombin

Abstract Bovine prothrombin has been purified by modifications of existing procedures. The purified protein is effectively homogeneous in four analytical gel electrophoresis systems, and is stable during storage in 50% glycerol-H2O at -20°. The specific activity of the protein when assayed by a modified two-stage procedure is 1,100 to 1,300 NIH thrombin units per mg. The molecular weight of the proenzyme determined by gel filtration in both 6 m guanidinium chloride, and electrophoresis in sodium dodecyl sulfate is 72,000 ± 5,000. The activation of prothrombin in 25% sodium citrate-defibrinated plasma has been studied by monitoring molecular weight changes and the production of enzymatic activity toward fibrinogen and N-α-tosyl-l-arginine methylester. The results of this study are consistent with a mechanism in which prothrombin is first cleaved to an intermediate of 65,000 daltons. This intermediate is subsequently cleaved to produce two single chain molecules of 39,000 daltons and 24,000 daltons. The 39,000-dalton chain is thrombogenic, giving rise to an active thrombin composed of two disulfide-linked chains (33,000 and 6,000 daltons). Further cleavages in this molecule result in two 28,000-dalton thrombins; one composed of two disulfide-linked chains (18,000 and 10,000 daltons) and the other composed of three disulfide-linked chains (14,000, 4,000, and 10,000 daltons). The thrombins produced in the activation system are chromatographically and electrophoretically indistinguishable from those in Parke-Davis topical thrombin.