Two Forms of Human Thrombin

Abstract

Abstract When preparations of human prothrombin are rapidly activated to thrombin (EC 3.4.4.13) in the presence of a partially purified enzyme from Taipan snake venom (Oxyuranus scutellatus scutellatus), the thrombin activity isolated from the activation mixture by ion exchange chromatography is associated with a single molecule of molecular weight of approximately 39,000. The subunit structure of this blood-clotting enzyme was examined by sodium dodecyl sulfate electrophoresis and was found to consist of two polypeptide chains connected by disulfide bonds, of molecular weight of approximately 32,000 and 7,000. NH2-terminal amino acid analysis by the 5-dimethylaminonaphthalene-1-sulfonyl procedure indicated that the larger chain contained NH2-terminal isoleucine while the smaller chain contained NH2-terminal threonine. With time, the enzyme self-digests itself without change in the clotting activity of the system to produce a smaller thrombin molecule of molecular weight of approximately 26,000, which consists of a 19,000 and a 7,000 molecular weight peptide with NH2-terminal isoleucine and threonine, respectively. The other peptide, which is inactive against fibrinogen, is split from the larger chain of the parent molecule presumably because of autolysis. It was found to have a molecular weight of approximately 11,000 and to contain a single NH2-terminal glycine. These structural changes were confirmed by isolation of the various thrombin peptides by gel filtration in 10% acetic acid. Slow and incomplete activation of human prothrombin in the presence of concentrated citrate solution or activation with a human Factor Xa preparation results in the isolation of a mixture of both thrombin molecules from the activation systems, including the inactive thrombin peptide. These molecules showed the same sequence of structural changes due to autolysis without a corresponding change in clotting activity as did the venom-derived enzyme. Thrombin preparations containing a mixture of the larger and smaller molecules, particularly those in which the smaller molecule predominates, have a significantly higher clotting activity than preparations containing only the larger molecule. No evidence was obtained for the existence of a three-chain polypeptide human thrombin molecule as has been reported by others for the bovine enzyme. Since preliminary observations seemed to indicate that under our conditions of study bovine and horse thrombin preparations contain similar species of molecules as those described above for human thrombin, methods of preparation of prothrombin or activation (or both) employed by others may account for this apparent discrepancy. These studies suggest that the thrombin derived from human, bovine, and possibly horse prothrombin appear quite similar in size, NH2-terminal amino acids, and subunit structure, their relative composition in a prothrombin activation mixture being dependent only upon the rate or yield (or both) of thrombin generated from its zymogen, rather than the source of activators.