COAGULATION. 457 



slaughter-house is washed thoroughly in running water until the 

 hemoglobin is removed, and is then minced and extracted at a low 

 temperature with the strong salt solution for several days. The 

 filtered extract is rich in thrombin, but contains also large amounts 

 of dissolved protein. Starting with such an extract the author* 

 has shown that by repeated shakings with chloroform the coagu- 

 lable proteins present in the extract may be removed completely 

 and the thrombin, in diminished quantities, be left behind in 

 apparently pure condition. 



A simpler method of obtaining relatively pure preparations of thrombin 

 in permanent form for experimental work and demonstrations may be de- 

 scribed briefly as follows: 



1. Fibrin obtained from the slaughter-house is washed thoroughly to re- 

 move all hemoglobin. The mass is squeezed to remove excess of water, is 

 then cut into fine pieces with scissors, and covered with an 8 per cent, solution 

 of sodium chlorid. The mixture is left for forty-eight hours in a refrigerator, 

 with occasional stirring. 



2. The salt solution is drained off through cheese-cloth. 



3. The filtrate is precipitated by the addition of an equal volume of acetone, 

 and this mixture is filtered as quickly as possible through a number of small 

 filters, not using more than 50 c.c. to a filter. After the liquid has run through 

 the filters are opened, and the precipitate is spread as thin as possible with a 

 spatula. The filters are then dried rapidly in a current of cold air before an 

 electric fan. 



4. The dried filters may be kept indefinitely in a desiccator, and when 

 thrombin is needed may be extracted with a little water, or the thrombin may 

 be extracted from the whole mass by covering the filters with water, al- 

 lowing to stand for one or two hours, and then filtering without squeezing 

 the filters. The clear solution of fibrin thus obtained may be distributed in 

 watch glasses, 1 or 2 c.c. to a glass, and be dried in front of an electric fan. 

 These dried preparations may be kept indefinitely in a desiccator. When 

 needed the contents of each glass are dissolved in 2 or 3 c.c. of water or normal 

 saline. This preparation contains a little coagulable protein in addition to 

 thrombin. If desired this impurity may be removed by dialyzing and shaking 

 with chloroform, or by reprecipitation with acetone. 



Observations made upon preparations of thrombin purified 

 as just described show that it has the following properties: it is 

 very easily soluble in water, it is not coagulated by boiling, it is 

 precipitated with difficulty by alcohol in excess, it is precipitated 

 uninjured by half-saturation with ammonium sulphate, it responds 

 to a number of the ordinary protein tests, such as the biuret, the 

 Millon's, and especially the tryptophan (Adamkiewicz) reaction. 

 We may conclude, therefore, that in all probability thrombin is a 

 protein substance. The evidence at hand indicates that thrombin 

 as such does not exist in the circulating blood, but is present prob- 

 ably in an antecedent or inactive form known as prothrombin or 

 thrombogen. So far as is known this prothrombin is furnished only 

 by the blood-platelets. When the blood is shed, or under certain 

 abnormal conditions while circulating in the vessels, the pro- 



* Howell, "American Journal of Physiology," 26, 453, 1910. 



