On the occurrence of polymorphic human prothrombin. Electrophoretic and chromatographic alterations of the molecule due to the action of thrombin.

Abstract This study was carried out to determine if previous reports by others concerning the polymorphic nature of prothrombin could be substantiated. The results obtained suggest that preparations of this important blood coagulation zymogen may undergo spontaneous activation with conversion to a very small but significant amount of thrombin. The active enzyme is then capable of producing cleavage at various points on the prothrombin molecule, which results in a heterogeneous mixture of molecules with different chromatographic and electrophoretic properties. Some of these molecules retain prothrombin activity. It seems probable that these events are responsible for the polymorphic nature of prothrombin previously reported, and they may also be indirectly responsible for the deleterious effects that certain cellulose and polymethacrylate ion exchange columns have been reported to produce during chromatography of the zymogen. A relatively homogeneous prothrombin preparation has been obtained by chromatography of less pure products on diethylaminoethyl cellulose columns at pH 7.4 with the use of a continuous buffer gradient. The zymogen thus isolated readily converts to thrombin in 25% (w/v) sodium citrate solution. However, if the preparations are altered owing to partial activation, this does not occur as readily but requires many days for the maximum yield of thrombin to be attained. Polyacrylamide disc electrophoresis, column chromatography, and gel filtration analysis have permitted the identification and isolation of at least seven activation fragments present in the prothrombin conversion system. They appear qualitatively and, in some respects, quantitatively in relation to the thrombin activity developed. These are compared to previous studies of the sedimentation and enzymatic properties of the prothrombin-thrombin system.