Is Thrombin an Enzyme?

Abstract

The earlier work on blood clotting, from the time of Schmidt, 1 was done mainly on the assumption that thrombin was an enzyme (fibrin ferment). The first seemingly definite evidence that it was not an enzyme was offered by Rettger 2 and later by the more careful work of Howell. 3 These 2 authors presented data showing a rather definite combining ratio between thrombin and blood fibrinogen when minimal amounts of the former were present. Howell raised a doubt as to the interpretation of the data, however, when he described the formation of repeated crops of fibrin in the serum following the removal of the fibrin masses, upon the weight of which they had based their first conclusions. He himself likened this behavior of thrombin to an enzyme, but still leaned toward the belief that it probably was not such. We have extended Howell's observations and obtained striking results which we briefly summarize below.

To fresh, clear, citrated dog plasma was added (NH₄)₂SO₄ sufficient to give 1/7 saturation and the precipitated fibrinogen filtered off, repeatedly washed in 20% NaCl, dissolved in 0.5% sodium citrate solution, reprecipitated with MgSO₄, washed and finally redissolved in 0.5% sodium citrate solution again. To the plasma, after the removal of the fibrinogen, (NH₄)₂SO₄ was further added to ½ saturation, and the precipitate collected and pressed free of liquid on a filter. This precipitate was stirred into 30% MgSO₄ solution, filtered, and the filtrate saturated with MgSO₄. The precipitate formed in this last step was collected and pressed free of as much liquid as possible. On treatment with cephalin and calcium it yielded very active thrombin, which deteriorated only slightly during 5 weeks of observation. The portion of the (NH₄)₂SO₄ precipitate that was insoluble in 30% MgSO₄ was found to contain little prothrombin.