Esterase and Clotting Activities Derived from Citrate Activation of Human Prothrombin

Abstract

Abstract When human prothrombin is activated in 25% (weight per volume) sodium citrate, the apparent clotting and esterase activities of thrombin develop at equal rates and reach maximum levels in about 16 hours. Following this, the esterase activity remains stable, but over 90% of the fibrinogen-clotting activity disappears from the system during the next 500 hours. Chromatography of the 16-hour system yields a single protein peak containing 40% of the protein and all of the clotting and esterase activity of the activation system. Chromatography of the 500-hour system yields two protein peaks, the major one having essentially only esterase activity and a minor one which has principally clotting activity. This results in almost a 2-fold purification of the esterase fraction over that obtained by chromatography of the 16-hour system. Comparisons were made between the preparations having both clotting and esterase activity and those having only esterase or clotting activity. With respect to the elution volume on gel filtration, electrophoresis, and sedimentation properties, no differences were observed. All preparations behaved as essentially single components having an estimated molecular weight of 30,000 g, with no evidence of dissociation to enzymatically active smaller molecules. Titration experiments with phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate showed a striking difference between the preparations, however. In addition, the clotting preparation had a profound affect on the citrate activation of prothrombin, while the esterase preparation did not. The evidence thus obtained indicates that thrombin is similar to certain other estero-proteolytic enzyme systems in which the molecule can be modified so as to alter its relative activity toward ester and protein substrates. This is discussed in relation to various interpretations concerning the nature of the active center on the thrombin molecule, as well as the implications these findings may have on the factors believed necessary for the inception of blood coagulation.